Cell processing using magnetic particles

ABSTRACT

The present invention relates to compositions comprising magnetic particles, the methods of using these compositions in processing animal sperm, the resulting sperm and embryo products, and the methods of use of these compositions to increase the efficiency, efficacy and/or speed of cell processing and artificial insemination techniques.

The present invention relates to compositions comprising magneticparticles, the methods of using these compositions in processing animalsperm, the resulting sperm and embryo products, and the methods of useof these compositions to increase the efficiency, efficacy and/or speedof cell processing and artificial insemination techniques.

BACKGROUND

Assisted reproductive technology (ART) includes such techniques as invitro fertilization (IVF), artificial insemination (AI),intracytoplasmic sperm injection (ICSI) (other techniques usingenucleated cells) and multiple ovulation and embryo transfer (MOET) (aswell as other embryo transfer techniques) and is used across the animalkingdom. ART methods are generally expensive, time consuming andmarginally successful given the inherent fragility of gametes whenoutside of their natural environments. Furthermore, the use of ARTwithin the animal breeding industry in a commercially feasible manner isadditionally challenging due to the limited availability of geneticallydesirable gametes (sperm or oocytes). One way to lower the cost of ARTand to improve its commercial feasibility is to increase the efficiencyof the involved processes by improving the viability and overall qualityof gametes used in ART. Although there has been a growing interest inthis field over the course of the last decade or so, there still remainsa strong need to increase the overall quality of gametes for use in ART,especially when breeding focuses on pre-natal gender selection,including improving gametes viability, motility and fertility, as wellas other longevity characteristics.

For example, in conventional AI, one problem limiting its commercialapplication in certain species is the need to use extremely high numberof sperm per AI dose to ensure successful fertilization currently. Inswine in particular, the need for improved sperm quality is especiallystrong since the typical dose of boar sperm required for successfulfertilization using conventional artificial insemination techniques,such as intra-cervical insemination, is currently 1×10⁹ sperm to 3×10⁹sperm.

Processing gametes such as flushed oocytes or sperm, both conventionaland sex sorted, before their use in ART may add a tremendous amount ofstress on the gamete cell(s) and often negatively impacts their cellularintegrity and membrane structure, which in turn may be reflected indecreased viability, motility and fertility. An example of processinggametes prior to their use in ART is the sorting of sperm based on sex(known as “gender enrichment” or “sex sorting”), which is a now commonlyused procedure to minimize wasted births of the wrong sex for selectivebreeding in the livestock industry. In some species, however, it isstill cost prohibitive and can represent a financial risk to those withsmaller breeding herds. Sex sorting includes processes that physicallyseparate X and Y bearing sperm from each other into separatesubpopulations, as well as processes in which sperm bearing theundesired sex chromosome in a sperm sample are selectively killed,compromised, disabled, rendered immotile, or otherwise renderedinfertile by, for example, laser ablation/photo damage techniques torender a gender enriched population of sperm.

The sex sorting process severely stresses and damages the cells andproduces a low percentage of useful sperm, which although capable offertilizing matured oocytes, may have reduced viability, motility andfertility compared to unprocessed cells. Typically, sex sorting involvesmany harsh steps including but not limited to: the initial collectionand handling of sperm ejaculate, which naturally starts to deterioraterapidly upon collection; the staining of sperm, which involves bindingof an excitable dye to the DNA or a harmful membrane selectionprocedure; the physical sorting of the sperm using high energyfluorescence that physically energizes the dye that is bound to the DNA,forced orientation through a narrow orifice, and application of anelectrical charge to the cell; the physical collection of the cells intoa receiving container, which often shocks the fragile cell upon contact;the osmotic stresses associated with dilution of the sperm droplet incollection media; and the storage of the sorted sperm usually byfreezing, which is well known to raise havoc with the cell's membranesystems. Each step places the processed sperm under abnormal stress thatdiminishes the overall motility, viability and/or fertility of thesperm. The result can lead to less efficient samples for use in ART,such as IVF and AI, and other types of subsequent or further processing.

Even non-sorted processed sperm exhibits significant losses infertility, viability and motility when being collected, handled andtransported without freezing, and noticeably experiences significantstress when mixed with cryoprotectant and frozen and thawed. Many in thefield have tried to improve methods for the use on unsorted,conventional semen to minimize loss in the handling processes associatedwith in vitro handling, preservation and use of semen samples.

Regardless of the processing, sperm lose their potential to fertilizewhen exposed to: elevated temperatures, abnormal buffers, stains,altered pH systems, physical pressurized orientation as when forcedthrough a nozzle or when oscillated to form drops in a flow cytometer,radiation used to illuminate the DNA binding dye, physical stressorsassociated with separation and collection techniques, cryoprotectants,freezing, thawing and micromanipulation by the handler.

There remains a continuing need to improve current methods of ART toreduce the cost and to make the procedures more dependable, efficient,fast and efficacious and commercially feasible to those on a restrictedbudget, especially for smaller breeders for whom sex-selection breedingmay be a high risk and expensive option. Accordingly, there is asignificant need for improved sperm processing techniques.

SUMMARY OF THE INVENTION

A broad object of the present invention is to provide improvements inthe motility, viability, fertility and overall integrity of processedsperm, particularly sperm that undergo analysis and/or sex sorting. Inorder to achieve such improvements, one aspect of the present inventionbroadly encompasses compositions comprising magnetic particles and theuse of such compositions in the cell sorting process. Other aspects ofthe invention encompass methods of using sperm processed with magneticparticles in various ART procedures, including but not limited to, IVF,AI, ICSI and MOET.

In one embodiment, the invention comprises a composition for magneticcellular manipulation comprising a plurality of particles. Each particlein the plurality of particles may include a magnetic substrate. Themagnetic substrate may be characterized by a magnetic susceptibilitygreater than zero. Such particles are generally referred to hereinafteras particles or magnetic particles. Each particle in the plurality ofparticles may also include a chargeable silicon-containing compound. Thechargeable silicon-containing compound may coat at least a portion ofthe magnetic substrate.

In another embodiment, a method for magnetic cellular manipulation isprovided. The method may include contacting a composition with abiological sample to form a mixture. The composition may include aplurality of particles. Each particle in the plurality of particles mayinclude a magnetic substrate. The magnetic substrate may becharacterized by a magnetic susceptibility greater than zero. Eachparticle in the plurality of particles may include a chargeablesilicon-containing compound. The chargeable silicon-containing compoundmay coat at least a portion of the magnetic substrate. The biologicalsample may include cells, such as sperm and/or cellular structures. Themethod may also include applying a magnetic field to the mixture tomanipulate the composition.

In one embodiment, a kit for magnetic cellular manipulation is provided.The kit may include instructions. The instructions may includecontacting a composition with a biological sample to form a mixture. Theinstructions may also include applying a magnetic field to the mixtureto manipulate the composition. The kit may also include the composition.The composition may include a plurality of particles. Each particle inthe plurality of particles may include a magnetic substrate. Themagnetic substrate may be characterized by a magnetic susceptibilitygreater than zero. Each particle in the plurality of particles mayinclude a chargeable silicon-containing compound. The chargeablesilicon-containing compound may coat at least a portion of the magneticsubstrate.

In certain embodiments of the invention, compositions comprisingmagnetic particles comprise sperm at a concentration of 500×10⁶ cells/mlor less; 400×10⁶ cells/ml or less; 300×10⁶ cells/ml or less; 200×10⁶cells/ml or less; 180×10⁶ cells/ml or less; 160×10⁶ cells/ml or less;120×10⁶ cells/ml or less; 100×10⁶ cells/ml or less; or 50×10⁶ cells/mlor less.

In other embodiments of the invention, compositions comprising magneticparticles comprise 0.01-3 mg of magnetic particles per 100 millionsperm; 0.1-2 mg of magnetic particles per 100 million sperm; 0.1-2 mg ofmagnetic particles per 100 million sperm; 0.2-1.8 mg of magneticparticles per 100 million sperm; 0.5-1.6 mg of magnetic particles per100 million sperm; 0.7-1.5 mg of magnetic particles per 100 millionsperm; 1.0-1.4 mg of magnetic particles per 100 million sperm; 1.1-1.3mg of magnetic particles per 100 million sperm; or 1.125 mg of magneticparticles per 100 million sperm.

In some embodiments of the invention, the magnetic particles have a sizedistributions from about 300 nm to about 800 nm; about 50 nm to about400 nm; about 1 nm to about 1 μm; from about 10 nm to about 900 nm; fromabout 10 nm to about 700 nm; from about 10 nm to about 500 nm; fromabout 10 nm to about 400 nm; from about 10 nm to about 300 nm; fromabout 10 nm to about 250 nm; from about 10 nm to about 200 nm; fromabout 10 nm to about 190 nm; from about 10 nm to about 180 nm; fromabout 10 nm to about 150 nm; from about 20 nm to about 700 nm; fromabout 20 nm to about 500 nm; from about 20 nm to about 400 nm; fromabout 20 nm to about 300 nm; from about 20 nm to about 250 m; from about20 nm to about 200 nm; from about 20 nm to about 190 nm; from about 20nm to about 180 nm; from about 20 nm to about 150 nm; from about 30 nmto about 500 nm; from about 30 nm to about 400 nm; from about 30 nm toabout 300 nm; from about 30 nm to about 250 m; from about 30 nm to about200 nm; from about 30 nm to about 190 nm; from about 30 nm to about 180nm; from about 30 nm to about 150 nm. In a further embodiment of theinvention, the magnetic particles exclude particles having a size of 200nm.

In certain embodiments of the invention, sex sorting of sperm may beaccomplished using any process or device known in the art for cellanalysis, sorting and/or population enrichment including but not limitedto use of a flow cytometer or use of a microfluidic chip. Ascontemplated herein, sex sorting in addition to encompassing techniquesfor physically separating, or isolating, X and Y bearing sperm from eachother as with droplet sorting and fluid switching sorting, alsoencompasses techniques for gender enrichment in which sperm bearing theundesired sex chromosome are killed, immobilized, or otherwise renderedinfertile, such as by use of laser ablation/photo damage techniques.

In one embodiment of the invention, compositions comprising magneticparticles may comprise one or more buffers, including but not limited tocarbonates, phosphates, citrates, acetates, lactates, and combinationsthereof, or a solution containing a salt, a carbohydrate, or acombination thereof can be employed in some of the embodiments of theinvention, such as, but not limited to, Tris, TES, HEPES, TALP, TCA,PBS, citrate, milk and derivatives thereof, as discussed in detail inU.S. Pat. No. 7,208,265 the contents of which is hereby incorporated byreference in its entirety.

In certain embodiments, compositions comprising magnetic particles maycomprise one or more chelators, including but not limited to,deferoxamine, deferasirox, penicillamine, alpha lipoic acid, DMPS, DMSA,dimercaprol and aminopolycarboxylic acids (complexones), including butnot limited to Fura-2, IDA, NTA, EDTA, DTPA, EGTA, BAPTA, NOTA, DOTA andnicotianamine, and derivatives thereof.

In certain embodiments, compositions comprising magnetic particles maycomprise low sugar media. The term “sugar” as used herein refers tomono- or di-saccharides that are generally metabolized by mammaliansperm, e.g., glucose and fructose. The term “sugar additive” as usedherein means sugar that is added to a media as a discrete compound andnot as a naturally occurring component of another additive in the mediasuch as egg yolk, seminal fluid or milk.

In certain embodiments of the invention, low sugar media comprises lessthan about 50 mM, 45 mM, 40 mM, 35 mM, 30 mM, 25 mM, 20 mM, 19 mM, 18mM, 17 mM, 16.5 mM, 16 mM, 15.5 mM, 15 mM, 14.5 mM, 14 mM, 13.5 mM, 13mM, 12.5 mM, 12 mM, 11.5 mM, 11 mM, 10.9 mM, 10.8 mM, 10.7 mM, 10.6 mM,10.5 mM, 10.4 mM, 10.3 mM, 10.2 mM, 10.1 mM, 10.0 mM, 10 mM, 9.75 mM,9.5 mM, 9.25 mM, 9.0 mM, 9 mM, 5 mM of sugar additive. In otherembodiments of the invention, low sugar media comprises about 1-5 mM,5-10 mM, 10-15 mM, 15-20 mM, 20-25 mM, 25-30 mM, 35-40 mM, or 45-50 mMof sugar additive. In further embodiments of the invention, low sugarmedia comprises about 1-5 mM, 1-10 mM, 1-15 mM, 1-20 mM, 1-25 mM, 1-30mM, 1-35 mM, 1-40 mM, 1-45 mM, or 1-50 mM of sugar additive. In yetfurther embodiments of the invention, low sugar media comprises about0.1 ppm to about 5 mM, about 0.1 ppm to about 10 mM, about 0.1 ppm toabout 15 mM, about 0.1 ppm to about 20 mM, about 0.1 ppm to about 25 mM,about 0.1 ppm to about 30 mM, about 0.1 ppm to about 35 mM, about 0.1ppm to about 40 mM, about 0.1 ppm to about 45 mM, or about 0.1 ppm toabout 50 mM of sugar additive. In other embodiments of the invention,low sugar media comprises about 1 mM, 2 mM, 3 mM, 4 mM, 4.1 mM, 4.2 mM,4.3 mM, 4.4 mM, 4.5 mM, 4.6 mM, 4.7 mM, 4.8 mM, 4.9 mM, 5 mM, 5.0 mM,5.1 mM, 5.2 mM, 5.3 mM, 5.4 mM, 5.5 mM, 5.6 mM, 5.7 mM, 5.8 mM, 5.9 mM,6 mM, 7 mM, 8 mM, 9 mM, 9.1, 9.2, 9.3, 9.4, 9.5, 9.6, 9.7, 9.8, 9.9, 10mM, 10.0, 10.1, 10.2, 10.3, 10.4, 10.5, 10.6, 10.7, 10.8, 10.9, 11 mM,12 mM, 13 mM, 14 mM, 15 mM, 16 mM, 17 mM, 18 mM, 19 mM, 20 mM, 25 mM, 30mM, 35 mM, 40 mM, 45 mM, or 50 mM of sugar additive. In a furtherembodiment of the invention, low sugar media comprises about 1-10 mM,2-9 mM, 3-6 mM, 4-6 mM, or 4-5 mM of sugar additive. In yet anotherembodiment of the invention, low sugar media comprises about 5-15 mM,6-14 mM, 7-13 mM, 8-12 mM, 9-11 mM or 9-10 mM of sugar additive. In yetanother embodiment of the invention, low sugar media comprises no sugaradditive or at most, trace amounts of sugar additive (i.e., no more than0.1-20 ppm of sugar additive).

In certain embodiments of the invention, low sugar media comprises lessthan about 50 mM, 45 mM, 40 mM, 35 mM, 30 mM, 25 mM, 20 mM, 19 mM, 18mM, 17 mM, 16.5 mM, 16 mM, 15.5 mM, 15 mM, 14.5 mM, 14 mM, 13.5 mM, 13mM, 12.5 mM, 12 mM, 11.5 mM, 11 mM, 10.9 mM, 10.8 mM, 10.7 mM, 10.6 mM,10.5 mM, 10.4 mM, 10.3 mM, 10.2 mM, 10.1 mM, 10.0 mM, 10 mM, 9.75 mM,9.5 mM, 9.25 mM, 9.0 mM, 9 mM, 5 mM of sugar. In other embodiments ofthe invention, low sugar media comprises about 1-5 mM, 5-10 mM, 10-15mM, 15-20 mM, 20-25 mM, 25-30 mM, 35-40 mM, or 45-50 mM of sugar. Infurther embodiments of the invention, low sugar media comprises about1-5 mM, 1-10 mM, 1-15 mM, 1-20 mM, 1-25 mM, 1-30 mM, 1-35 mM, 1-40 mM,1-45 mM, or 1-50 mM of sugar. In yet further embodiments of theinvention, low sugar media comprises about 0.1 ppm to about 5 mM, about0.1 ppm to about 10 mM, about 0.1 ppm to about 15 mM, about 0.1 ppm toabout 20 mM, about 0.1 ppm to about 25 mM, about 0.1 ppm to about 30 mM,about 0.1 ppm to about 35 mM, about 0.1 ppm to about 40 mM, about 0.1ppm to about 45 mM, or about 0.1 ppm to about 50 mM of sugar. In otherembodiments of the invention, low sugar media comprises about 1 mM, 2mM, 3 mM, 4 mM, 4.1 mM, 4.2 mM, 4.3 mM, 4.4 mM, 4.5 mM, 4.6 mM, 4.7 mM,4.8 mM, 4.9 mM, 5 mM, 5.0 mM, 5.1 mM, 5.2 mM, 5.3 mM, 5.4 mM, 5.5 mM,5.6 mM, 5.7 mM, 5.8 mM, 5.9 mM, 6 mM, 7 mM, 8 mM, 9 mM, 9.1, 9.2, 9.3,9.4, 9.5, 9.6, 9.7, 9.8, 9.9, 10 mM, 10.0, 10.1, 10.2, 10.3, 10.4, 10.5,10.6, 10.7, 10.8, 10.9, 11 mM, 12 mM, 13 mM, 14 mM, 15 mM, 16 mM, 17 mM,18 mM, 19 mM, 20 mM, 25 mM, 30 mM, 35 mM, 40 mM, 45 mM, or 50 mM ofsugar. In a further embodiment of the invention, low sugar mediacomprises about 1-10 mM, 2-9 mM, 3-6 mM, 4-6 mM, or 4-5 mM of sugar. Inyet another embodiment of the invention, low sugar media comprises about5-15 mM, 6-14 mM, 7-13 mM, 8-12 mM, 9-11 mM or 9-10 mM of sugar. Inanother embodiment of the invention, low sugar media comprises no sugaror at most, trace amounts of sugar (i.e., no more than 0.1-20 ppm ofsugar).

In other embodiments, compositions comprising magnetic particles maycomprise one or more “organic stress reducing” agents (OSRs), which maycomprise an antioxidant, a vitamin or other organic molecule involveddirectly or indirectly in modulating physiological stresses in the cell.In certain embodiments, compositions comprising magnetic particles maycomprise one or more OSRs, each in the concentration range of 0.01 mg/mlto 5 mg/ml. Various OSRs can be used in the context of the currentinvention, including but not limited to: catalase, superoxide dismutase(SOD), SOD mimics, glutathione, glutathione reductase, glutathioneperoxidase, pyruvate, mercaptoethanol, butylated hydroxytoluene (BHT),lipoic acid, flavins, quinones, vitamin K (and related vitamers),vitamin B12 (and related vitamers), with ‘vitamers’ defined as compoundshaving the same vitamin activity (such as cobalamin, cyanocobalamin,methylcobalamin, adenosylcobalamin, hydroxocobalamin, and pseudo-B12),vitamin E (including its vitamers, tocopherols (α, β, γ), tocotrienols,and α-tocopheryl), alpha-ketoglutarate (also known as α-KG, AKG oroxo-glutarate) and various biological forms of AKG (such as arginine,aspartate, lysine, and similar derivatives), other compounds thatregulate nitric oxide in the cell including malondialdehyde (MDA) andasymmetric dimethylarginine (ADMA), and biologically active derivativesthereof.

Certain embodiments of the invention utilize concentrations of OSRsselected from the following ranges: 0.01 to 5.0 mg/ml; 0.01 to 0.25mg/ml; 0.01 to 0.5 mg/ml; 0.01 to 1 mg/ml; 0.01 to 2.5 mg/ml; 0.01 to 5mg/ml; 0.05 to 0.1 mg/ml; 0.05 to 1.0 mg/ml; 0.05 to 2.5 mg/ml; 0.1 to0.25 mg/ml; 0.1 to 0.5 mg/ml; 0.1 to 1 mg/ml; 0.1 to 2.5 mg/ml; 0.1 to 5mg/ml; 0.15 to 0.45 mg/ml; 0.15 to 0.5 mg/ml; 0.25 to 0.35 mg/ml; 0.25to 0.5 mg/ml; 0.25 to 1 mg/ml; 0.25 to 2.5 mg/ml; 0.25 to 5 mg/ml; 0.35to 0.5 mg/ml; 0.35 to 1 mg/ml; 0.35 to 2.5 mg/ml; 0.35 to 5 mg/ml; 0.5to 1 mg/ml; 0.5 to 2.5 mg/ml; 0.5 to 5 mg/ml; 1 to 2.5 mg/ml; 1 to 5mg/ml; about 0.05 mg/ml; about 0.1 mg/ml; about 0.15 mg/ml; about 0.25mg/ml; about 0.35 mg/ml; about 0.45 mg/ml; and about 0.5 mg/ml.

In other embodiments, compositions comprising magnetic particles maycomprise one or more tricarboxylic acid cycle intermediates, includingbut not limited to, pyruvate, acetyl-CoA, citrate, isocitrate,α-ketoglutarate, succinyl-CoA, succinate, fumarate, malate,oxaloacetate, and derivatives thereof, including but not limited toisomers and acids. In a particular embodiment, compositions comprisingmagnetic particles comprises two or more tricarboxylic acid cycleintermediates, including but not limited to, pyruvate, acetyl-CoA,citrate, isocitrate, α-ketoglutarate, succinyl-CoA, succinate, fumarate,malate, oxaloacetate, and derivatives thereof, including but not limitedto isomers and acids.

Certain embodiments of the invention utilize concentrations of atricarboxylic acid cycle intermediate selected from the followingranges: 0.01 to 5.0 mg/ml; 0.01 to 0.25 mg/ml; 0.01 to 0.5 mg/ml; 0.01to 1 mg/ml; 0.01 to 2.5 mg/ml; 0.01 to 5 mg/ml; 0.05 to 0.1 mg/ml; 0.05to 1.0 mg/ml; 0.05 to 2.5 mg/ml; 0.1 to 0.25 mg/ml; 0.1 to 0.5 mg/ml;0.1 to 1 mg/ml; 0.1 to 2.5 mg/ml; 0.1 to 5 mg/ml; 0.15 to 0.45 mg/ml;0.15 to 0.5 mg/ml; 0.25 to 0.35 mg/mi; 0.25 to 0.5 mg/ml; 0.25 to 1mg/ml; 0.25 to 2.5 mg/ml; 0.25 to 5 mg/ml; 0.35 to 0.5 mg/ml; 0.35 to 1mg/ml; 0.35 to 2.5 mg/ml; 0.35 to 5 mg/ml; 0.5 to 1 mg/ml; 0.5 to 2.5mg/ml; 0.5 to 5 mg/ml; 1 to 2.5 mg/ml; 1 to 5 mg/ml; about 0.05 mg/ml;about 0.1 mg/ml; about 0.15 mg/ml; about 0.25 mg/ml; about 0.35 mg/ml;about 0.45 mg/ml; and about 0.5 mg/ml.

In other embodiments, compositions comprising magnetic particles maycomprise one or more cryoprotectants, including but not limited to,propylene glycol, dimethyl sulfoxide, ethylene glycol and glycerol, or acombination thereof. In certain embodiments, compositions comprisingmagnetic particles may comprise a concentration of cryoprotectant bypercent volume selected from the following: less than 1%; 1-5%; 5%; 5 to10%; 10%; 10 to 20%; 16.7%; 20%; 20 to 30%; or 30 to 40%.

In a further embodiment, compositions comprising magnetic particles maycomprise one or more antioxidants, including but not limited tocatalase, superoxide dismutase (SOD), SOD mimics, glutathione,glutathione reductase, glutathione peroxidase, pyruvate,mercaptoethanol, butylatedhydroxytoluene (BHT), lipoic acid, flavonoids,phenolic acids and their esters, quinines, vitamin A (and relatedvitamers), vitamin C (and related vitamers), vitamin K (and relatedvitamers), vitamin B12 (and related vitamers), with “vitamers” definedas compounds having the same vitamin activity (such as cobalamin,cyanocobalamin, methylcobalamin, adenosylcobalamin, hydroxocobalamin,and pseudo-B12), vitamin E (including its vitamers, tocopherols (α, β,γ), tocotrienols, and α-tocopheryl), α-ketoglutarate (also known asα-KG, AKG or oxo-glutarate) and various biological forms of AKG (such asarginine, aspartate, lysine, and similar derivatives), coenzyme Q,manganese, iodide, melatonin and carotenoid terpenoids.

Certain embodiments of the invention utilize concentrations of anantioxidant selected from the following ranges: 0.01 to 5.0 mg/ml; 0.01to 0.25 mg/ml; 0.01 to 0.5 mg/ml; 0.01 to 1 mg/ml; 0.01 to 2.5 mg/ml;0.01 to 5 mg/ml; 0.05 to 0.1 mg/ml; 0.05 to 1.0 mg/ml; 0.05 to 2.5mg/ml; 0.1 to 0.25 mg/ml; 0.1 to 0.5 mg/ml; 0.1 to 1 mg/ml; 0.1 to 2.5mg/ml; 0.1 to 5 mg/ml; 0.15 to 0.45 mg/ml; 0.15 to 0.5 mg/ml; 0.25 to0.35 mg/ml; 0.25 to 0.5 mg/ml; 0.25 to 1 mg/ml; 0.25 to 2.5 mg/ml; 0.25to 5 mg/ml; 0.35 to 0.5 mg/ml; 0.35 to 1 mg/ml; 0.35 to 2.5 mg/ml; 0.35to 5 mg/ml; 0.5 to 1 mg/ml; 0.5 to 2.5 mg/ml; 0.5 to 5 mg/ml; 1 to 2.5mg/ml; 1 to 5 mg/ml; about 0.05 mg/ml; about 0.1 mg/ml; about 0.15mg/ml; about 0.25 mg/ml; about 0.35 mg/ml; about 0.45 mg/ml; and about0.5 mg/ml.

In yet another embodiment, compositions comprising magnetic particlesmay comprise one or more protein sources, including but not limited to,egg yolk, egg yolk extract, milk (including heat homogenized and skim),milk extract, soy protein, soy protein extract, serum albumin, bovineserum albumin, human serum substitute supplement, seminal proteins, suchas, for example, whole seminal plasma or seminal plasma extracts, andderivatives thereof. In certain embodiments, compositions comprisingmagnetic particles may comprise a concentration of protein source bypercent volume selected from the following: 1-5%; 5%; 5 to 10%; 10%; 10to 20%; 16.7%; 20%; 20 to 30%; or 30 to 40%.

In a further embodiment of the invention, compositions comprisingmagnetic particles may comprise one or more antimicrobial or antibioticagents, including but not limited to, tylosin, gentamicin, lincomycin,spectinomycin, Linco-Spectin® (lincomycin hydrochloride-spectinomycin),penicillin, streptomycin, ticarcillin, polymyxin B, and theirderivatives. If included, the antibiotics may be present in aconcentration of about 50 μg to about 800 μg per ml of semen. In anotherembodiment compositions comprising magnetic particles may comprise adetergent, including but not limited to, an alkyl ionic detergent suchas sodiumdodecyl sulfate (SDS).

In another embodiment, compositions comprising magnetic particles maycomprise one or more salts, including but not limited to, NaCl, KCl,MgCl₂, CaCl₂) and any combination of a salt-forming anion, including butnot limited to acetate (CH₃COO⁻), carbonate (CO₃ ²⁻), chloride (Cl⁻),citrate (HOC(COO⁻)(CH2COO⁻)₂), fluoride (F⁻), nitrate (NO₃ ⁻), nitrite(NO₂ ⁻), phosphate (PO₄ ³⁻) and sulfate (SO₄ ²⁻) and a salt-formingcation, including but not limited to, ammonium (NH₄ ⁺), calcium (Ca²⁺),iron (Fe²⁺ and Fe³⁺), magnesium (Mg²⁺), potassium (K⁺), pyridinium(C₅H₅NH⁺), quaternary ammonium NR₄ ⁺ and sodium (Na⁺).

In yet another embodiment, compositions comprising magnetic particlesmay comprise one or more growth factors including but not limited totransforming growth factors (“TGF”), such as, for example, TGFβ-1 andTGFβ-2, and insulin-like growth factors (“IGF”), such as for example,IGF-1.

Another embodiment of the invention encompasses a staining solutioncomprising magnetic particles and a DNA selective dye. DNA selectivedyes for use with the invention include but are not limited to UV lightexcitable, selective dyes, such as Hoechst 33342 and Hoechst 33258 andvisible light excitable dyes, such as SYBR®-14 and bisbenzimide-BODIPY®conjugate6-{[3-((2Z)-2-{[1-(difluoroboryl)-3,5-dimethyl-1H-pyrrol-2yl]methylene}-2H-pyrrol-5-yl)propanoyl]amino}-N-[3-(methyl{3-[({4-[6-(4-methylpiperazin-1-yl)-1H,3′H-2,5′bibenzimidazol-2′-yl]phenoxy}acetyl)amino]propyl}amino)propyl]hexanamide.Each of these dyes may be used alone or in combination. In anotherembodiment, the staining solution further comprises one or more dyequenchers, including but not limited to F&DC red food dye No. 40 andyellow food dye No. 4. In yet another embodiment, the staining solutionfurther comprises one or more OSRs.

In one embodiment of the invention, the concentration of dye in thestaining solution is from about 0.1 μM to about 1.0 μM; from about 0.1μM to about 1000 μM; from about 100 μM to about 500 μM; from about 200μM to about 500 μM; from about 300 μM to about 450 μM; about 350 μM;about 400 μM; or about 450 μM.

The pH of compositions comprising magnetic particles of the inventionmay be maintained at any of a range of pHs suitable for the particularprocess and sperm type. In certain embodiments, this will be in therange of about 5.0 to about 9.0; in the range of 5.5 to about 7.8; fromabout 5.0 to about 7.0; from about 6.0 to about 7.0; from about 6.0 toabout 6.5; from about 6.0 to about 8.0; from about 6.5 to about 7.5;from about 6.8 to about 7.4; from about 7.0 to about 9.0; from about 7.0to about 8.0; from about 7.0 to about 7.5; about 6.2, about 6.5; about6.6; about 6.7; about 6.8; about 6.9; about 7.0; about 7.1; about 7.2;about 7.3; about 7.35; about 7.4; or about 7.5; about 7.6, about 7.7,about 7.8, about 7.9; or about 8.0.

In certain embodiments, magnetic particles may be combined with a spermsample to form a sperm composition. The term “sperm sample” may comprisea processed semen sample or an unsorted, conventional semen sample. Insome embodiments of the invention, the sperm composition comprisingmagnetic particles is used to process sperm for use in ART. Such ARTtechniques involve different levels of gamete cell processing which inthe case of sperm can entail, by example only and is not limited to oneor more of the following: artificially collecting a semen sample fromthe male animal that may involve natural, electronic or other types ofsexual stimulation; holding; transporting; buffering with different pHs;chilling; warming; staining; diluting; concentrating; energeticallyexciting as with a laser; electronic charging; deflecting; ablating tokill unwanted cells usually with targeted lasers; sorting; collecting;shaking; oscillating; magnetically separating; oxygenating as associatedwith microchip sorting procedures; labeling; precipitating;centrifuging; resuspending; mixing; dialyzing; cryostabilizing;freezing; vitrifying; cryopreserving; thawing; culturing; inseminating;microinjecting; microfluidic processing; microchip processing; jet andair processing; flow cytometry processing; and similar handlingtechniques. Whereas a single processing step may exert only minimalstress on a sperm, others or a combination may add significant stress,often killing the cell. An example is the sex sorting process used toseparate X- from Y-chromosome bearing cells; the sorting processcombines a large number of independent stressful steps that maycompromise the overall integrity of the sorted sperm population.

In some embodiments of the invention, a sperm composition comprising asperm sample and magnetic particles can be used immediately or processedwithin the first few minutes after the magnetic particles are added tothe sperm sample for whatever processing step is needed, whereby theholding period would be in the range of 1 to 2 seconds; 1 to 10 seconds;1 to 20 seconds; 20 to 30 seconds; 30 seconds to 1 minute; 1 to 2minutes; 1 second to 3 minutes; 1 minute to 5 minutes; or 1 minute to 10minutes. In other embodiments, holding periods can be longer, as in therange of a 1 minute to 15 minutes, the range of 15 minutes to 1 hour orup to about 8 hours or overnight for extensive processing such as withsex sorting techniques.

In some embodiments of the invention, magnetic particles are usedseveral times during a complex processing procedure. In otherembodiments, magnetic particles are used only at one or more particularsteps. By way of example, magnetic particles can be used during thestaining process during sex sorting, which is often performed atnon-physiological pH and at elevated temperatures. Similarly, magneticbeads can be used just prior to cryopreservation of sperm or afterthawing of cryopreserved sperm.

A further embodiment of the present invention provides a method ofimproving the motility, viability and/or fertility of a sperm samplethat has already undergone a sorting process, including but not limitedto sex sorting, comprising the step of contacting or adding a sortedsperm sample to magnetic particles and then subsequently removing themagnetic particles.

Another broad object of the present invention is to improve themotility, viability (including longevity and ability to surviveenvironmental stress) and fertility of processed and/or sorted sperm foruse in ART such as IVF, AI, ICSI (as well as other techniques usingenucleated cells), and MOET (as well as other embryo transfertechniques). Some embodiments of the invention encompass compositionscomprising magnetic particles comprising a sorted or processed spermsample, and optionally at least one OSR in the range of 0.01 mg/ml to 5mg/ml, for use in ART.

A further embodiment of the invention resides in a method of making anembryo comprising mixing at least one egg with one or more sperm from asperm sample that has been contacted with magnetic particles. Theembryos produced by this method constitute a further embodiment of theinvention.

Another embodiment of the invention includes a method for inseminatingan organism through an AI technique using a processed or sorted spermsample contacted with magnetic particles. The progeny of the organismthat results from the aforementioned AI method also constitutes anembodiment of the invention. A further embodiment of the inventionencompasses a method for recovering embryos that are produced from theaforementioned AI method.

Embodiments of the invention can include sperm, or spermatozoa,collected from numerous species of male animals, and the inventionshould be understood not to be limited to the species of male animalsdescribed by the specific examples within this application. Rather thespecific examples within this application are intended to beillustrative of the varied and numerous species of male animals fromwhich semen can be collected and utilized in certain embodiments of theinvention. Embodiments of the invention, for example, may include thesperm of humans as well as animals having commercial value for meat ordairy production such as swine, ovine, bovine, equine, deer, elk,buffalo, or the like (naturally the mammals used for meat or dairyproduction may vary from culture to culture). It may also include thesperm of various domesticated mammalian species encompassed by caninesand felines, as well as sperm of primates, including but not limited tochimpanzees, gorillas, or humans and the spermatozoa from whales,dolphins and other marine mammals. It may also include frozen-thawedsperm from all the various mammals above-described and further,including but not limited to, the sperm of deceased donors, from rare orexotic mammals, zoological specimens, or endangered species.

A particular embodiment of the invention comprises a method of sorting asperm sample to form one or more subpopulations comprising the steps ofproviding a sperm sample, sorting the sperm sample to form one or moresubpopulations and using magnetic particles during one or more of theaforementioned sorting steps. In the context of sorting sperm using aflow cytometer, for example, magnetic particles may be used in a diluentfor diluting sperm, a staining solution for staining the sperm with, forexample, a DNA selective dye, a sheath fluid for encapsulating the corestream containing the sperm as it passes through the flow cytometer, acatch media for receiving one or more of the sorted spermsubpopulations, or a resuspension media for resuspending processedsperm.

Another embodiment of the invention encompasses a composition comprisinga gender enriched sperm population, created for example by way ofseparation or photo/laser ablation, and magnetic particles.

Another embodiment of the invention encompasses a method of processingsperm comprising the steps of forming a composition comprising saidsperm and magnetic particles; removing said magnetic particles from saidcomposition; forming a stream comprising said sperm; determining aproperty of said sperm in said stream; and selecting sperm having aproperty of interest from said sperm in said stream. In a furtherembodiment, the steps of forming a composition comprising said sperm andmagnetic particles and removing said magnetic particles from saidcomposition are performed after the step of selecting sperm having aproperty of interest from said sperm in said stream. In yet anotherembodiment, the steps of forming a composition comprising said sperm andmagnetic particles and removing said magnetic particles from saidcomposition are performed before the step of forming a stream comprisingsaid sperm. In a further aspect of the invention, magnetic particlesthat have been removed from a composition comprising sperm are washedone or more times in a suitable media in order to extract any viableand/or uncompromised sperm that may have adhered to the particles.

BRIEF DESCRIPTION OF THE DRAWINGS

In order that the invention may be more clearly understood, variousembodiments of the present invention will now be described by way ofexample only with reference to the accompanying sheets of drawingswherein:

FIG. 1 is a schematic representation of part of a flow cytometerillustrating a method of sorting a sperm sample into one or moresubpopulations according to some embodiments of the present invention.

FIG. 2 shows the results of ICP and Zeta analysis of multiple lots ofmagnetic particles.

FIG. 3 shows various sort parameters and percentage of dead spermremoved by magnetic particles in Example 2.

FIG. 4 shows the percentage of dead sperm removed by magnetic particlesin Example 2.

FIG. 5 shows post-thaw motility and viability of sperm treated withmagnetic particles in Example 2.

FIG. 6 shows various sort parameters and percentage of dead spermremoved by sonicated and non-sonicated magnetic particles in Example 2

FIG. 7 shows and the percentage of dead sperm removed by sonicated andnon-sonicated magnetic particles in Example 2.

FIG. 8 shows post-thaw motility and viability of sperm treated withsonicated and non-sonicated magnetic particles in Example 2.

FIG. 9 is a flow cytometer image indicating the percentage of dead spermremoved by magnetic particles.

FIG. 10 shows various sort parameters and percentage of dead spermremoved by magnetic particles in Example 5.

FIG. 11 shows the percentage of dead sperm removed by magnetic particlesin Example 5.

FIG. 12 shows 3 hour post-thaw motility of sperm treated with magneticparticles in Example 5.

FIG. 13 shows 0 hour post-thaw motility of sperm treated with magneticparticles in Example 5.

FIG. 14 shows the average 0 and 3 hour post-thaw motility of spermtreated with magnetic particles in Example 5.

FIG. 15 shows various sort parameters and percentage of dead spermremoved by magnetic particles in Example 6.

FIG. 16 shows the average percentage of dead sperm removed by magneticparticles in Example 6.

FIG. 17 shows 0 and 3 hour post-thaw motility of sperm treated withmagnetic particles in Example 6.

FIG. 18 shows the average 0 and 3 hour post-thaw motility of spermtreated with magnetic particles in Example 6.

FIG. 19 shows quality control analysis results for 8 bulls used inExample 7.

FIG. 20 shows the percentage of dead sperm removed by magnetic particlesin Example 7.

FIG. 21 shows the average sort and abort rates achieved in Example 7

FIG. 22 shows the percentage of dead sperm removed by magnetic particlesin Example 7.

FIG. 23 shows post-thaw motility and viability of sperm treated withmagnetic particles in Example 7.

FIG. 24 shows IVF trial results using sperm treated with magneticparticles in Example 7.

FIG. 25 shows Zeta potential distribution form magnetic particlesproduced according to Example 9.

FIG. 26 shows the percentage of oriented sperm and the percentage ofdead sperm in Example 10.

FIG. 27 shows the percentage of dead sperm removed by magnetic particlesin Example 10.

FIG. 28 shows the percentage of dead sperm removed by magnetic particlesin Example 11.

FIG. 29 shows various sort parameters and percentage of dead spermremoved by magnetic particles in Example 12.

FIG. 30 shows the percentage of dead sperm removed by magnetic particlesin Example 12.

FIG. 31 shows the percentage of dead sperm removed by magnetic particlesin Example 13.

FIG. 32 shows the percentage of dead sperm removed by magnetic particlesin Example 13.

FIG. 33 shows the percentage of dead sperm removed by magnetic particlesin Example 14.

FIG. 34 shows the percentage of dead sperm removed by magnetic particlesin Example 14.

FIG. 35 shows various sort parameters in Example 15.

FIG. 36 shows the percentage of dead sperm removed by magnetic particlesin Example 15.

FIG. 37 shows post-thaw motility and viability of sperm treated withmagnetic particles in Example 15.

FIG. 38 shows various sort parameters and percentage of dead spermremoved by magnetic particles in Example 15.

FIG. 39 shows the percentage of dead sperm removed by magnetic particlesin Example 15.

FIG. 40 shows the percentage of dead sperm removed by magnetic particlesin Example 15.

FIG. 41 shows post-thaw motility and viability of sperm treated withmagnetic particles in Example 15.

FIG. 42 shows IVF trial results using sperm treated with magneticparticles in Example 15.

DETAILED DESCRIPTION OF THE INVENTION

The invention broadly encompasses methods and compositions comprisingmagnetic particles for processing sperm. It has been discovered that theuse of magnetic particles for processing sperm improves the relativenumber of viable and/or uncompromised sperm relative to dead and/orcompromised sperm in a sample, as well as the overall quality of theprocessed sperm, including but not limited to increased motility,viability and fertility. Furthermore, by decreasing the number of deadand/or compromised sperm in a sample prior to sex sorting, the overallspeed and efficiency of the sex sorting process can be increased. Thenet result is higher quality, lower cost gender enriched sperm that canbe used in ART.

Briefly described, embodiments of the described methods and processesinclude a method for removing or identifying cells or cellularstructures having damaged membranes from those with intact membranes,thereby enriching sample, or cellular, viability and quality. The methodmay be applied to cells such as contained in freshly collected samples,after dilution, during and after cooling, or during and after other cellor system procedures that may be employed prior to, or after,cryopreservation, or to frozen/thawed cell samples. The method may alsobe used for samples that may be used immediately. The method may also beused for samples that may be held for a period of time or extended inbuffers or other substances. For example, the method may also be usedfor samples that may be held at 4° C. to 40° C. for at least about 12,24, 30, 36, 48, 60, 72 hours or more. The method may also be used forsamples that include but are not limited to samples that have anosmolarity of 250-375 mOsm. The enriched cell populations may be usedfor routine procedures, prior to or after other processing techniques,prior to or after shipment of samples, and prior to or after long-termcryopreservation or other processes.

Embodiments related to sperm may include a method for removing spermhaving damaged membranes from those with intact membranes to enrichsperm viability of a sperm sample. The method may be applied to spermcontained in freshly collected neat ejaculates, after dilution, duringand after cooling, or during and after other semen processing proceduresthat may be employed prior to, or after, cryopreservation, or tofrozen/thawed sperm. The method may also be used for neat or extendedsperm samples to be used immediately. The method may also be used forneat or extended sperm samples held up to 30 h at 4° C. to 40° C., orextended in sperm rich buffers that have an osmolarity of 250-375 mOsm.The enriched sperm populations may be used for routine artificialinsemination, prior to or after sperm sexing techniques, prior to orafter shipment of semen for routine or sperm sexing purposes orcryopreservation purposes, or for in vitro fertilization, for allmammalian sperm.

In some embodiments, damage to the membranes of intact cells may bereduced by removing known harmful effects caused by damaged cells. Forexample, DNA fragmentation, oxidative damage caused by peroxidation, andthe premature release of proteolytic and hydrolytic enzymes may beexamples of effects attributable to membrane damage. Damage to cellsample integrity may reduce lifespan both in vitro and in vivo, mayreduce desired cellular functional ability, and may cause poor resultantcapabilities.

With respect to sperm as one, non-limiting example, damage to themembranes of intact sperm may be reduced by removing known harmfuleffects caused by damaged sperm. For example, DNA fragmentation,oxidative damage caused by peroxidation, and the premature release ofproteolytic and hydrolytic enzymes may be examples of sperm damagecaused by membrane damaged sperm. Damage to spermatozoal integrity mayreduce sperm lifespan both in vitro and in vivo, may reducefertilization ability, and likely causes poor embryo quality, which maybe a major source of infertility in mammals.

Mammalian sperm with good fertility may exhibit a high frequency ofmorphologically-normal, viable sperm. Current procedures for semenprocessing for sex selection, cooling, or cryopreservation may havedetrimental effects on the metabolism and motility of sperm, as well ason the status of sperm membrane domains. The net result of these effectsmay be reduced sperm functionality. Magnetic removal of damaged orcompromised cells or cellular structures may reduce a detrimental effecton the quality of live and normal sperm that may be caused by dead andabnormal sperm.

In various embodiments, the described processes and methods may be usedto differentiate necrotic, apoptotic, and normal cells. In the examplediscussed, the carboxyl modified silane surface binds to the membrane ofthe dead and dying sperm through an electrical charge interaction knownas zeta potential. Material may spontaneously acquire a positive ornegative surface electrical charge when brought into contact with apolar medium (e.g., water). For example, an interface in deionized watermay be negatively charged. An ionization of surface groups to form asurface electrical charge may be observed with metal oxide surfaces(M—OH) as well as materials that may contain carboxyl and/or aminogroups, such as proteins, ionic polymers, and polyelectrolytes.Ionization and/or dissociation, degree of charge development, netmolecular charge, and sign, either positive or negative, may depend onthe pH of the surrounding medium.

The conjugation of carboxyl group functional magnetic particles mayadditionally be applied to, for example, fluorescent stains. SYBR®-14and bis-benzamide are example membrane permeable stains that may be usedto distinguish cells from other background substances. In the sperm cellembodiment, such fluorescent stains may distinguish sperm cells fromdiluent particles frequently found in extenders employed in non-frozenstorage or cryopreservation of sperm. Other examples of fluorescentprobes may include JC-1 and rhodamine 123, which may be used to assessthe respiration rate of cell mitochondria; or fluorescently labeledagglutins from the pea (PSA) or peanut (PNA) that may be used to detectacrosome-reacted cells such as sperm. Other labels include but are notlimited to acridine orange (e.g., to remove apoptotic cells);7-aminoactinomycin D (7-AAD), which may be also a DNA intercalatingagent in double stranded DNA with a high affinity for GC rich regions;food coloring such as Allura Red (FD&C Red #40), Sunset Yellow (FD&CYellow #6), Indigo carmine (FD&C Blue #2), and Fast Green FCF (FD&C #3).

In some embodiments of the described methods and processes, cells withvarying degrees of membrane damage may be labeled with magneticparticles containing a charged surface. This may be in contrast to theuse of annexin-V/microbead magnetic cell sorting procedures that fail toidentify and/or remove pre-capacitated or acrosomal reacted spermbecause the PS does not become externalized in these examples. Whenmembrane damaged sperm or cellular structures labeled with the surfacecharged magnetic particles are placed in a magnetic field, such cells orcellular structures may be eliminated from the general population. Theresultant harvested sub-population of viable cells, perhaps such assperm, may be further processed for cryopreservation, non-frozentransport and storage, functional utilization (such as sex selection forsperm), or used in related aspects, perhaps such as assistedreproductive technologies (ARTs) for sperm or the like.

Embodiments of the described methods and processes may be used with anytype of magnetically identifying separating apparatus, including but notlimited to devices incorporating columns, such as magnetic-activatedcell sorting (MACS) products, devices using simple magnetic fieldsapplied to test tubes or containers, or high throughput magneticdevices.

Targeted dead and dying cells labeled with magnetic particles andsubjected to magnetic cell separation in an open, column-less magneticsystem may be removed more efficiently and in greater numbers per timeunit compared to flow cytometry. Magnetic cell separation may beutilized with no internal operating pressure, or if pressurized, a lowerinternal operating pressure; and the stream of fluid containing thecells may avoid being broken into cell damaging droplets as for flowcytometry. Further, the sheath fluid for flow cytometry may be generallya salt-based, lipoprotein-deficient physiological medium. Magnetic cellseparation may allow some cells, such as sperm, to be bathed innutrient-rich buffers that may promote and prolong cell viability duringthe separation procedure.

The described methods and processes may remove necrotic cells orcellular structures that have been traumatized during cell processingprocedures such as cryopreservation, centrifugation, and staining.Necrotic damage may occur by different cellular processes than thatcaused in one example by sperm senescence, which may be a naturallyoccurring cause of cellular death.

In other magnetic cell separation applications, embodiments of thedescribed methods and processes may be used to label cells uniquely,such as sperm, with one or more fluorochrome stains, targeting aspecific cell or sperm attribute. The targeted cell or sperm cell may beselectively killed or rendered non-functional, with an energy source,including but not limited to an electrical charge or pulse of laserlight. The described methods and processes may be used to magneticallylabel and ultimately remove the non-functional cell or sperm throughmagnetic cell separation procedures. The resultant desiredsub-population of harvested cells may be selected for membraneintactness (viability) as well as for specific cellular attributes,including but not limited to, in the sperm example, chromosomal sexselection.

In various embodiments, a composition for magnetic cellular manipulationis provided. The composition may include a plurality of particles. Eachparticle in the plurality of particles may include a magnetic substrate.The magnetic substrate may be characterized by a magnetic susceptibilitygreater than zero. Each particle in the plurality of particles may alsoinclude a chargeable silicon-containing compound. The chargeablesilicon-containing compound may coat at least a portion of the magneticsubstrate.

“Magnetic susceptibility” means the response of a sample, such as themagnetic substrate, to an externally applied magnetic field. Forexample, a magnetic susceptibility of less than or equal to zero may beassociated with diamagnetism. A magnetic susceptibility of greater thanzero may be associated with magnetic properties other than diamagnetism.For example, in various embodiments, the magnetic substrate may becharacterized by one or more of paramagnetism, superparamagnetism,ferromagnetism, or ferrimagnetism. The magnetic substrate may include ametal oxide, such as a transition metal oxide, for example, an ironoxide. In some examples, the magnetic substrate may include Fe₃O₄.

A “chargeable silicon-containing compound” is any silicon containingmolecule, polymer, or material that may be caused to acquire or hold acharge, e.g., via functionalization with charged or chargeable moieties.Chargeable/charged moieties may include, but are not limited to, species(and ions thereof) of: metals; oxides; carboxylates; amines; amides;carbamides; sulfates; sulfonates; sulfites; phosphonates; phosphates;halides; hydroxides; and combinations thereof. For example, thechargeable silicon-containing compound may include2-(carbomethoxy)ethyltrimethoxysilane.

In various examples, the composition may include a zeta potentialcharge. For example, the chargeable silicon-containing compound mayinclude a negative zeta potential charge. The chargeablesilicon-containing compound may include a positive zeta potentialcharge. In some examples, at least a portion of the plurality ofparticles may include a first zeta potential charge. The portion of theplurality of particles may form a complex with one or more cells orcellular structures that include a second zeta potential charge. Thesecond zeta charge may be opposite in sign compared to the first zetacharge. In several embodiments, at least a portion of the plurality ofparticles may include a negative zeta potential charge. The portion ofthe plurality of particles may form a complex with one or more spermcells or sperm cellular structures that include a positive zetapotential charge.

In another embodiment, a method for magnetic cellular manipulation isprovided. The method may include contacting a composition with abiological sample to form a mixture. The composition may include aplurality of particles. Each particle in the plurality of particles mayinclude a magnetic substrate. The magnetic substrate may becharacterized by a magnetic susceptibility greater than zero. Eachparticle in the plurality of particles may include a chargeablesilicon-containing compound. The chargeable silicon-containing compoundmay coat at least a portion of the magnetic substrate. The biologicalsample may include cells and/or cellular structures. The method may alsoinclude applying a magnetic field to the mixture to manipulate thecomposition.

A “biological sample” may include any natural or prepared compositionthat includes the cells and/or cellular structures. Natural samples mayinclude, for example, biological fluids containing cells or cellularstructures, such as blood, lymphatic fluids, intestinal fluids,intercellular fluids, sweat, tears, urine, semen, mucosal secretions,synovial fluid, and the like. Natural samples may include fluidstypically free of cells or cellular structures, but which may includecells or cellular structures as part of injury, illness, genetic defect,or other pathological conditions. Prepared samples may include anybiopsy, tissue homogenate, or other prepared form of biological tissue.Typically, the biological sample will include at least one cell orcellular structure characterized by a zeta potential charge. Thebiological sample may include at least two or more cells or cellularstructures characterized by zeta potential charges differing in sign orcharge density. For example, a biological sample may include a firstcell characterized by a first zeta potential charge and a second cellcharacterized by a second zeta potential charge opposite in sign to thefirst zeta potential charge.

In some embodiments, the method may include causing the chargeablesilicon-containing compound to acquire a first zeta potential charge.The first zeta potential charge may be opposite in sign compared to asecond zeta potential charge comprised by the cells and/or cellularstructures in the biological sample. Causing the chargeablesilicon-containing compound to acquire the first zeta potential chargemay include contacting the chargeable silicon-containing compound to apolar medium, as described herein.

In several embodiments, the method may include causing the compositionand at least a portion of the cells and/or cellular structures in thebiological sample to form a complex. Applying the magnetic field to themixture to manipulate the composition may manipulate the complex.

In some embodiments, at least a portion of the plurality of particlesfurther comprises at least one of a protein, an antibody, and a dye.

In several embodiments, the biological sample may include viable cellsand damaged or compromised cells or cellular structures. The compositionmay selectively form a complex with one of the viable cells or thedamaged or compromised cells or cellular structures, for exampleaccording to a first zeta potential charge on the composition and anopposite second zeta potential charge on one of the viable cells or thedamaged or compromised cells or cellular structures. The method mayfurther include separating the viable cells from the damaged orcompromised cells or cellular structures by applying the magnetic fieldto the mixture. Because the composition may selectively form a complexwith one of the viable cells or the damaged or compromised cells orcellular structures, the portion of the biological sample forming thecomplex with the composition may be magnetically manipulated andseparated from portions of the biological sample not forming the complexwith the composition. The method may therefore be a method forselectively and magnetically separating portions of the biologicalsample according to zeta potential charge.

In various embodiments, the biological sample may include viable spermcells and damaged or compromised sperm cells or sperm cellularstructures. The composition may form a complex with the damaged orcompromised sperm cells or sperm cellular structures. The method mayfurther include separating the viable sperm cells from the complexincluding the damaged or compromised sperm cells or sperm cellularstructures by applying the magnetic field to the mixture. Because thecomposition may selectively form a complex with the damaged orcompromised sperm cells or sperm cellular structures, the complex withthe damaged or compromised sperm cells or sperm cellular structures maybe magnetically manipulated and separated from the viable sperm cells.The method may therefore be a method for selectively and magneticallyseparating viable sperm cells from the damaged or compromised spermcells or sperm cellular structures according to zeta potential charge.

In several embodiments, the method may include subjecting the spermsample to detection, for example fluorescence detection as describedherein.

In various embodiments, a kit for magnetic cellular manipulation isprovided. The kit may include instructions. The instructions may includecontacting a composition with a biological sample to form a mixture. Theinstructions may also include applying a magnetic field to the mixtureto manipulate the composition. The kit may also include the composition.The composition may include a plurality of particles. Each particle inthe plurality of particles may include a magnetic substrate. Themagnetic substrate may be characterized by a magnetic susceptibilitygreater than zero. Each particle in the plurality of particles mayinclude a chargeable silicon-containing compound. The chargeablesilicon-containing compound may coat at least a portion of the magneticsubstrate.

In some embodiments of the kit, the biological sample may include viablecells and damaged or compromised cells or cellular structures. Thecomposition may form a complex with one of the viable cells or thedamaged or compromised cells or cellular structures. The instructionsmay further include separating the viable cells from the damaged orcompromised cells or cellular structures by applying the magnetic fieldto the mixture.

In several embodiments of the kit, the composition may be configured forforming a complex with damaged or compromised sperm cells or spermcellular structures. The instructions may further include selecting thebiological sample comprising viable sperm cells and damaged orcompromised sperm cells or sperm cellular structures. The instructionsmay also include separating the viable sperm cells from the complexincluding the damaged or compromised sperm cells or sperm cellularstructures by applying the magnetic field to the mixture.

Processing steps to which sperm are commonly subjected, and with whichthe invention may be used before, during or after, include, but are notlimited to, collecting from a male animal, which may involve natural,electronic or other types of sexual stimulation; holding; transporting;buffering; chilling; warming; staining; diluting; concentrating;energetically exciting (as with a laser, for example); electroniccharging; deflecting; ablating to kill unwanted cells usually withtargeted lasers; sorting; collecting; shaking; oscillating; magneticallyseparating; oxygenating as associated with microchip sorting procedures;labeling; precipitating; centrifuging; resuspending; mixing; dialyzing;cryostabilizing; freezing; vitrifying; cryopreserving; thawing;culturing; inseminating; microinjecting; microfluidic processing;microchip processing; jet and air processing; flow cytometry processing;and similar handling techniques.

Regardless of how sperm are to be ultimately utilized, the initialprocessing step is typically collection of a sperm sample from a male.Generally, the sperm sample is collected into an extender or diluentdesigned to sustain the cells until further processing or use.Alternatively, semen is collected and then subsequently diluted with anextender after collection. One embodiment of the invention encompassesprocessing collected semen with magnetic particles during or subsequentto collection.

Whereas a single processing step, such as collection, may exert onlyminimal stress on sperm, others or a combination may add significantstress, often killing the cells. An example is the sex sorting processused to separate X- from Y-chromosome bearing cells; the sorting processcombines a large number of independent stressful steps that compromisethe overall integrity of the sorted sperm population. Accordingly, in aparticular embodiment of the invention, compositions comprising magneticparticles are used in the sorting process, including but not limited tothe staining solution, sheath fluid and catch media, and prior to andafter cryopreservation of such processed cells.

I. Collecting Sperm

It is contemplated that intact viable bovine, porcine, equine, ovine,cervine, murine or other mammalian sperm, may be collected and contactedwith magnetic particles. Various methods of collection of viable spermare known and include, for example, the gloved-hand method, use of anartificial vagina, and electro-ejaculation. As an example, a bovinesperm sample, typically containing about 0.5 to about 10 billion spermper milliliter, may be collected directly from the source mammal, orfrom more than one source mammal of the same species, into a vesselcontaining magnetic particles to form a sperm composition. The spermcomposition may optionally comprise one or more OSRs, which may bepresent as constituents of the composition comprising magnetic particlesprior to contacting with the sperm, or which may be added to the spermcomposition, each OSR in the concentration range of 0.01 mg/ml to 5mg/ml. The magnetic particles may be subsequently removed from the spermcomposition prior to any further processing steps.

Once the sperm composition is in the laboratory, various quality checkscan be conducted, including checking the motility (e.g., via CASASystem), viability (e.g., via flow cytometer), morphology (e.g., viamicroscopy) and concentration (e.g., via NucleoCounter). Spermcompositions that pass these quality checks can then be prepared forfurther processing, such as sorting. A comparison of viewing chambersand slides can be done in a variety of IVOS instruments, which forexample only can be a Hamilton-Thorne IVOS (Hamilton-Thorne, Beverly,Mass.). Instrument settings may be set as follows: image capture; framesper second=60; number of frames=30; cell detection; minimum contrast=50;minimum cell size=5; defaults, cell size=5; cell intensity=50;progressive cells, path velocity=50 um/s; straightness ≥70%; slow cells(um/s); average path velocity (VAP, <30 um/s), straight-line velocity(VSL, <15 um/s). The CASA motility variables measured can be apercentage of total motile sperm (motile), percentage of progressivelymotile sperm (progressive), VAO, VSL, curvilinear velocity (VCL, um/s),average lateral head displacement (ALK, um) and the number of times thesperm head crosses the mean path/s (BCF, Hz), straight-line spermmotility (STR, %), and linear sperm motility (LIN, %). See for instance,Lenz, R W, et al., J AnimSci (2011) 89:383-388, incorporated byreference herein in its entirety.

Various OSRs can be used in the context of the current invention,including but not limited to catalase, superoxide dismutase (SOD), SODmimics, glutathione, glutathione reductase, glutathione peroxidase,pyruvate, mercaptoethanol, butylated hydroxytoluene (BHT), lipoic acid,flavins, quinines, vitamin K (and related vitamers), vitamin B12 (andrelated vitamers), with ‘vitamers’ defined as compounds having the samevitamin activity (such as cobalamin, cyanocobalamin, methylcobalamin,adenosylcobalamin, hydroxocobalamin, and pseudo-B12), vitamin E(including its vitamers, tocopherols (α, β, γ), tocotrienols, andα-tocopheryl), alpha-ketoglutarate (also known as α-KG, AKG oroxo-glutarate) and various biological forms of AKG (such as arginine,aspartate, lysine, and similar derivatives), other compounds thatregulate nitric oxide in the cell including malondialdehyde (MDA) andasymmetric dimethylarginine (ADMA); and biologically active derivativesthereof.

Alternatively, the semen sample may be collected into an empty vesseland then subsequently contacted with magnetic particles within severalminutes to hours after collection to form the sperm composition. Inaddition to a buffer, the sperm composition may also contain a range ofadditives, including but not limited to the aforementioned OSRs,chelators, tricarboxylic acid cycle intermediates, cryoprotectants,sterols, lipids, fatty acids, protein sources, antibiotics, growthfactors, caproic acid, catalase, Caprogen (caproic acid, catalase, and5% egg yolk) detergents, including alkyl ionic detergents such assodiumdodecyl sulfate (SDS). It should be noted that certain substancesmay be classified in one or more of the above listed categories ofadditives. For example, citrate may be considered both a tricarboxylicacid cycle intermediate and a buffer.

Exemplary buffers for use in the invention include, but are not limitedto, carbonates, phosphates, citrates, acetates, lactates, andcombinations thereof. Specific buffers that may be used include, but arenot limited to, Tris, TES, Pipes, HEPES, TALP, TCA, PBS, citrate, milkand derivatives thereof, which are discussed in detail in U.S. Pat. No.7,208,265 the contents of which is hereby incorporated by reference inits entirety.

Exemplary chelators for use in the invention include, but are notlimited to, deferoxamine, deferasirox, penicillamine, alpha lipoic acid,DMPS, DMSA, dimercaprol and aminopolycarboxylic acids (complexones),including but not limited to Fura-2, IDA, NTA, EDTA, DTPA, EGTA, BAPTA,NOTA, DOTA and nicotianamine, and derivatives thereof.

Exemplary tricarboxylic acid cycle intermediates for use in theinvention include, but are not limited to, pyruvate, acetyl-CoA,citrate, isocitrate, α-ketoglutarate, succinyl-CoA, succinate, fumarate,malate, oxaloacetate, and derivatives thereof, including but not limitedto isomers and acids. In a particular embodiment, magnetic particlescomprises two or more tricarboxylic acid cycle intermediates, includingbut not limited to, pyruvate, acetyl-CoA, citrate, isocitrate,α-ketoglutarate, succinyl-CoA, succinate, fumarate, malate,oxaloacetate, and derivatives thereof, including but not limited toisomers and acids.

Exemplary cryoprotectants for use in the invention include but are notlimited to propylene glycol, dimethyl sulfoxide, ethylene glycol andglycerol, or a combination thereof. In certain embodiments, magneticparticles may comprise a concentration of cryoprotectant by percentvolume (w/v) selected from the following: 1-5%; 5%; 5 to 10%; 10%; 10 to20%; 16.7%; 20%; 20 to 30%; or 30 to 40%.

Exemplary protein sources for use in the invention include egg yolk, eggyolk extract, milk (including heat homogenized and skim), milk extract,soy protein, soy protein extract, serum albumin, bovine serum albumin,human serum substitute supplement, seminal proteins, such as, forexample, whole seminal plasma or seminal plasma extracts (see, forexample, Parks et al., Sperm Membrane Phospholipid Peroxidation andFragmentation: Effects on Sperm Function and Role of Seminal PlasmaPAF-Acetylhydrolase, Proceedings of the 16th Technical Conference onArtificial Insemination & reproduction, 1996, the content of which ishereby incorporated herein by reference), and combinations thereof. Incertain embodiments, compositions comprising magnetic particles maycomprise a concentration of protein source by percent volume selectedfrom the following: 1-5%; 5%; 5 to 10%; 10%; 10 to 20%; 16.7%; 20%; 20to 30%; or 30 to 40%. Albumin, and more particularly bovine serumalbumin (BSA), is a commonly used protein source. For example, ifincluded, BSA may be present in the sperm composition in an amount of,less than about 5.0% (w/v); less than about 2% (w/v); less than about 1%(w/v); or about 0.1% (w/v).

The use of a protein source, such BSA, alone may initiate the process ofcapacitation in a percentage of the sperm in the composition. It isgenerally preferred that this process take place in the femalereproductive tract. Therefore, in order to inhibit the initiation ofcapacitation during dilution, as well as during subsequent processingstep such as staining and sorting, an alternative protein source or aprotein substitute may be included in the sperm composition. Thealternative protein source or protein substitute possess theadvantageous effects of a typical protein source, such as BSA, inaddition to the ability to inhibit the initiation of capacitation in alarger percentage of the cells in the sperm composition. Examples of aalternative protein sources includes human serum substitute supplement(SSS) (Irvine Scientific, Santa Ana, Calif.) and cholesterol enhancedBSA, while an example of a protein substitute includes a polyvinylalcohol, such as for example, a low to medium viscosity polyvinylalcohol generally of a molecular weight of about 30,000 to about 60,000.Generally, if included, these compositions will be present in the sameamounts as disclosed above with respect to BSA, with the total albumincontent of the buffer or buffered solution generally not exceeding about5.0% (w/v).

An antibiotic may be included in the sperm composition in order toinhibit bacterial growth. Exemplary antibiotics include, for example,tylosin, gentamicin, lincomycin, spectinomycin, Linco-Spectin®(lincomycin hydrochloride-spectinomycin), penicillin, streptomycin,ticarcillin, polymyxin B, or any combination thereof. If included, theantibiotics may be present in a concentration of about 50 μg to about800 μg per ml of semen, regardless of whether the semen is neat,buffered, or contains additional substances, such as for example, any ofthe additives mentioned herein. The Certified Semen Services (CSS) andNational Association of Animal Breeders (NAAB) have promulgatedguidelines regarding the use of antibiotics with respect to spermcollection and use.

A growth factor may be added to the sperm composition in order to helpmaintain the viability of the sperm. Exemplary growth factors include,for example, transforming growth factors (“TGF”), such as, for example,TGFβ-1 and TGFβ-2, and insulin-like growth factors (“IGF”), such as forexample, IGF-1. Generally, TGF may be present in the sperm compositionin the form of TGFβ-1 in a concentration of about 0.1 ng/L to about 10μg/L or as TGFβ-2 in a concentration of about 0.1 ng/L to about 200ng/L, and IGF may be present in the sperm composition in the form ofIGF-1 in a concentration of about 0.1 ng/L to about 50 μg/L. The use ofsuch growth factors is well known in the art and is disclosed, forexample, in U.S. Patent Application Publication No. 2003/0157473, thecontent of which is hereby incorporated herein by reference.

In certain embodiments of the invention, the collection fluid comprisessperm at a concentration of 200×10⁷ cells/ml or less; 100×10⁷ cells/mlor less; 500×10⁶ cells/ml or less; 400×10⁶ cells/ml or less; 300×10⁶cells/ml or less; 200×10⁶ cells/ml or less; 180×10⁶ cells/ml or less;160×10⁶ cells/ml or less; 120×10⁶ cells/ml or less; 100×10⁶ cells/ml orless; or 50×10⁶ cells/ml or less. Furthermore, in other embodiments ofthe invention, the staining solution comprises 0.01-3 mg of magneticparticles per 100 million sperm; 0.1-2 mg of magnetic particles per 100million sperm; 0.1-2 mg of magnetic particles per 100 million sperm;0.2-1.8 mg of magnetic particles per 100 million sperm; 0.5-1.6 mg ofmagnetic particles per 100 million sperm; 0.7-1.5 mg of magneticparticles per 100 million sperm; 1.0-1.4 mg of magnetic particles per100 million sperm; 1.1-1.3 mg of magnetic particles per 100 millionsperm; or 1.125 mg of magnetic particles per 100 million sperm.

Once collected, the sperm may be used, for example, in a stainingprocess, a sorting process, or a fertilization process. If magneticparticles are used in the collection fluid, that may be removed prior tofurther processing. It is contemplated that any such further use and/orprocessing of the sperm may utilize magnetic particles.

II. Sorting of Collected Sperm

A. Staining of the Cells

One embodiment of the invention encompasses the use of magneticparticles in a staining solution for sperm. A process of staining spermtypically comprises the formation of a staining solution containingintact viable sperm and a dye, sometimes referred to as a label. In thisaspect of the invention, the magnetic particles may be contacted withthe sperm to form a sperm composition, and then the sperm compositioncontacted with a DNA selective dye to form the staining solution.Alternatively, a DNA selective dye may be added to a magnetic particlesto form a staining solution, with sperm subsequently added to thestaining solution.

In this embodiment, the sperm source may be neat semen, oralternatively, a sperm-containing semen derivative obtained bycentrifugation or the use of other means to separate semen intofractions.

The pH of the staining solution may be maintained at any of a range ofpHs; typically this will be in the range of about 5.0 to about 9.0, orin the range of 5.5 to 7.8. The staining solution may be maintained at aslightly acid pH, i.e., from about 5.0 to about 7.0. Typically, the pHis from about 6.0 to about 7.0; from about 6.0 to about 6.5; about 6.2,about 6.5; about 6.6; about 6.7; about 6.8; about 6.9; or about 7.0.Alternatively, the staining solution may be maintained at a slightlybasic pH, i.e., from about 7.0 to about 9.0. Typically, the pH is about7.0 to about 8.0; about 7.0 to about 7.5; about 7.0; about 7.1; about7.2; about 7.3; about 7.35; about 7.4; or about 7.5.

The staining solution may be formed by using one or more UV or visiblelight excitable, DNA selective dyes as previously described in U.S. Pat.No. 5,135,759 and WO 02/41906, the contents of each of which are herebyincorporated herein by reference. Exemplary UV light excitable,selective dyes include Hoechst 33342 and Hoechst 33258. Exemplaryvisible light excitable dyes include SYBR®-14 and bisbenzimide-BODIPY®conjugate 6-{[3-((2Z)-2-{[1-(difluoroboryl)-3,5-dimethyl-1H-pyrrol-2yl]methylene}-2H-pyrrol-5-yl)propanoyl]amino}-N-[3-(methyl{3-[({4-[6-(4-methylpiperazin-1-yl)-1H,3′H-2,5′bibenzimidazol-2′-yl]phenoxy}acetyl)amino]propyl}amino)propyl]hexanamide(“BBC”) described in WO 02/41906. Each of these dyes may be used aloneor in combination; alternatively, other cell permeant UV and visiblelight excitable dyes may be used, alone or in combination with theaforementioned dyes, provided the dye does not detrimentally affect theviability of the sperm to an unacceptable degree when used inconcentrations which enable sorting or enrichment as describedelsewhere.

The staining solution may also comprise a dye quencher in addition to aDNA selective dye. Staining protocols for sex sorting, or even bulksorting, sperm typically rely upon the inclusion of F&DC red food dyeNo. 40 (“red food dye No. 40” or “red 40”) and/or yellow food dye No. 4as quenching dyes. The maximal absorbance wavelengths of these quenchingdyes overlaps the maximal emissions wavelengths of fluorescent dyes,including Hoechst 33342 when bound to nuclear or chromosomal DNA.Because red food dye No. 40 and yellow food dye No. 4 differentiallypermeate membrane-compromised sperm and overlap the emission spectra ofthe DNA selective fluorescent dye, FRET (florescence resonance energytransfer) between the light leaving the DNA-stain complex and the deadquenching dye reduces the overall detected intensity of the lightemitted from membrane compromised sperm. The quenched, or dampened,fluorescence from these cells provide fewer photons to the detectorsresulting in a distinctly lower signal. This distinctly lower signalresults in a noticeable separated subpopulation which allows theexclusion (“gating out”) of the membrane compromised sperm during thesorting procedure. Since membrane compromised sperm comprises largelynon-viable sperm, excluding these cells from the analysis results in anenriched sperm subpopulation with respect to viability in the sex sortedsubpopulation.

The staining solution may be formed using fluorescent polyamides, andmore specifically polyamides with a fluorescent label or reporterconjugated thereto. Such labels will fluoresce when bound to nucleicacids. Examples of polyamides with a fluorescent label or reporterattached thereto include, for example, those disclosed in Best et al.,Proc. Natl. Acad. Sci. USA, 15 100(21): 12063-12068 (2003); Gygi, etal., Nucleic Acids Res., 30(13): 2790-2799 (2002); U.S. Pat. Nos.5,998,140; 6,143,901; and 6,090,947, the content of each of which ishereby incorporated herein by reference.

Fluorescent nucleotide sequences may also be used to label the sperm.Such nucleotide sequences fluoresce when hybridized to a nucleic acidcontaining a target or complementary sequence, but are otherwisenonfluorescent when in a non-hybridized state. Such oligonucleotides aredisclosed, for example, in U.S. Patent Application Publication No.2003/0113765 (hereby incorporated herein by reference).

Sex specific antibodies may also be used to label the sperm in astaining solution. In this embodiment, for example, a sex specificantibody may be conjugated with a fluorescent moiety (or equivalentreporter molecule). Because the antibody binds to antigens present ononly an X chromosome-bearing or, alternatively, a Y chromosome-bearingcell, such cells can be selectively identified based upon theirfluorescence (versus the nonfluorescence of an unlabeled cell).Moreover, more than one sex specific antibody, each antibody having adifferent fluorescent moiety attached thereto, may be usedsimultaneously. This allows for differentiation of X chromosome-bearingand Y chromosome-bearing cells based upon the differing fluorescence ofeach.

Luminescent, color-selective nanocrystals may also be used to labelsperm in a staining solution. Also referred to as quantum dots, theseparticles are well known in the art, as demonstrated by U.S. Pat. Nos.6,322,901 and 6,576,291, each of which is hereby incorporated herein byreference. These nanocrystals have been conjugated to a number ofbiological materials, including for example, peptides, antibodies,nucleic acids, streptavidin, and polysaccharides, (see, for example,U.S. Pat. Nos. 6,207,392; 6,423,551; 5,990,479, and 6,326,144, each ofwhich is hereby incorporated herein by reference), and have been used todetect biological targets (see, for example, U.S. Pat. Nos. 6,207,392and 6,247,323, each of which is hereby incorporated herein byreference).

In certain embodiments of the invention, the staining solution comprisessperm at a concentration of 500×10⁶ cells/ml or less; 400×10⁶ cells/mlor less; 300×10⁶ cells/ml or less; 200×10⁶ cells/ml or less; 180×10⁶cells/ml or less; 160×10⁶ cells/ml or less; 120×10⁶ cells/ml or less;100×10⁶ cells/ml or less; or 50×10⁶ cells/ml or less. Furthermore, inother embodiments of the invention, the staining solution comprises0.01-3 mg of magnetic particles per 100 million sperm; 0.1-2 mg ofmagnetic particles per 100 million sperm; 0.1-2 mg of magnetic particlesper 100 million sperm; 0.2-1.8 mg of magnetic particles per 100 millionsperm; 0.5-1.6 mg of magnetic particles per 100 million sperm; 0.7-1.5mg of magnetic particles per 100 million sperm; 1.0-1.4 mg of magneticparticles per 100 million sperm; 1.1-1.3 mg of magnetic particles per100 million sperm; or 1.125 mg of magnetic particles per 100 millionsperm.

The concentration of the DNA selective or of any other type of dye inthe staining solution is a function of a range of variables whichinclude the permeability of the cells to the selected dye, thetemperature of the staining solution, the amount of time allowed forstaining to occur, the concentration of sperm, and the degree ofenrichment desired in the subsequent sorting or enrichment step. Ingeneral, the dye concentration is preferably sufficient to achieve thedesired degree of staining in a reasonably short period of time withoutsubstantially detrimentally affecting sperm viability. For example, theconcentration of Hoechst 33342, Hoechst 33258, SYBR®-14, or BBC in thestaining solution will generally be between about 0.1 μM and about 1.0M;from about 0.1 μM to about 1000 μM; from about 100 μM to about 500 μM;from about 200 μM to about 500 μM; or from about 300 μM to about 450 μM.Accordingly, under one set of staining conditions, the concentration ofHoechst 33342 is about 350 μM. Under another set of staining conditions,the concentration of Hoechst 33342 is about 400 μM. Under still anotherset of staining conditions the concentration is about 450 μM.

As another example, the concentration of a fluorescent polyamide, suchas for example, those described in U.S. Application Publication No.2001/0002314, will generally be between about 0.1 μM and about 1 mM;about 1 μM to about 1 mM; about 5 μM to about 100 μM; or about 10 μM.

Optionally, the staining solution may also contain additives to enhancesperm quality. Exemplary additives include one or more OSRs, anantibiotic, a growth factor or a composition which regulatesoxidation/reduction reactions intracellularly and/or extracellularly asdiscussed above with respect to cell sample collection. These additivesmay be added to the collection fluid in accordance therewith.

Once formed, the staining solution may be maintained at any of a rangeof temperatures; typically, this will be within a range of about 4° C.to about 50° C. For example, the staining solution may be maintained ata relatively low temperature, i.e., a temperature of about 4° C. toabout 30° C.; in this embodiment, the temperature is about 20° C. toabout 30° C.; from about 25° C. to about 30° C.; or about 28° C.Alternatively, the staining solution may be maintained within anintermediate temperature range, i.e., a temperature of about 30° C. toabout 39° C.; in this embodiment, the temperature is at about 34° C. toabout 39° C.; about 35° C.; or about 37° C. In addition, the stainingsolution may be maintained within a relatively high temperature range,i.e., a temperature of about 40° C. to about 50° C.; in this embodiment,the temperature is from about 41° C. to about 49° C.; from about 41° C.to about 45° C.; from about 41° C. to about 43° C.; or about 41° C.Selection of a preferred temperature generally depends upon a range ofvariables, including for example, the permeability of the cells to thedye(s) being used, the concentration of the dye(s) in the stainingsolution, the amount of time the cells will be maintained in thestaining solution, and the degree of enrichment desired in the sortingor enrichment step.

Uptake of dye by the sperm in the staining solution is allowed tocontinue for a period of time sufficient to obtain the desired degree ofDNA staining. That period is typically a period sufficient for the dyeto bind to the DNA of the sperm such that X and Y chromosome-bearingsperm may be sorted or enriched based upon the differing and measurablefluorescence intensity between the two. Generally, this will be no morethan about 24 hours; no more 30 than about 10 hours; no more than about2 hours; no more than about 90 minutes; no more than about 60 minutes;or from about 5 minutes to about 60 minutes. In a particular embodiment,the period is about 30 minutes or about 55 minutes.

The length of the staining period and the temperature at which stainingoccurs are related such that the longer the period of staining, thelower the temperature of staining temperature may be. For example, inone embodiment, the staining may occur at a relatively low temperatureand for a period of about 3 hours to about 24 hours. Alternatively, thestaining may occur at an intermediate temperature and for a period ofabout one half hour to about 3 hours. In addition, staining may occur ata relatively high temperature and for a period of about 10 minutes toabout 90 minutes. In a particular embodiment, staining may occur at atemperature of about 4° C. for a period of about 24 hours. In anotherembodiment, staining may occur at a temperature of about 18° C. for aperiod of about 4 hours. In yet another embodiment, staining may occurat a temperature of about 41° C. for a period of about 30 minutes. Inanother embodiment, staining may occur at a temperature of about 35° C.for a period of about 55 minutes. Accordingly, in one embodiment, astaining solution is formed comprising magnetic particles, sperm and adye in a concentration from about 100 μM to about 450 μM, and thestaining mixture is held for a period of time at a temperature of about28° C.; about 35° C.; or about 41° C. In another embodiment, the periodof time is about 30 minutes; about 55 minutes; or about 3 hours.

If magnetic particles are used in the staining solution, it iscontemplated that such magnetic particles may be removed prior to anyfurther processing steps. Alternatively, in another embodiment themagnetic particles may remain in the staining solution for furtherprocessing including sorting on a flow cytometer.

B. Sorting or Enriching of the Stained Sperm

Some embodiments include use of one or more OSRs as pre-mixed componentsof the prepared buffers, extenders, stains, catch fluids, and/orcryo-extenders used in the sex sorting procedure. In some cases, whenthe sorting of sperm is not going to involve sex sorting, a quenchingdye without the need for a DNA staining dye may be required, in whichcase the OSR will only be present with the quenching dye to form thestained sample. Commonly used and well known sorting methods includeflow cytometry systems, as exemplified by and described in U.S. Pat.Nos. 5,135,759, 5,985,216, 6,071,689, 6,149,867, and 6,263,745;International Patent Publications WO 99/33956 and WO 01/37655; and U.S.patent application Ser. No. 10/812,351 (corresponding InternationalPatent Publication WO 2004/088283), the content of each of which ishereby incorporated herein by reference. When sorting according to suchmethods, the sperm are introduced into the nozzle of a flow cytometer ina sample fluid. In one embodiment, the sample fluid may comprisemagnetic particles and the stained sperm.

As noted above, in certain embodiments of the invention, sex sorting ofsperm may be accomplished using any process or device known in the artfor cell analysis, sorting and/or population enrichment including butnot limited to use of a flow cytometer or use of a microfluidic chip,and encompasses techniques for physically separating X and Y bearingsperm from each other, as with droplet sorting and fluid switchingsorting, and techniques for gender enrichment in which sperm bearing theundesired sex chromosome are killed, immobilized, or otherwise renderedinfertile, such as by use of laser ablation/photo-damage techniques.

Generally, in certain embodiments, devices used with the inventiondetermine a property of sperm based on fluorescence emitted by the spermwhen passed before a source of illumination such as a laser beam. Thepresence or absence of an X- or Y-bearing chromosomes are examples ofsuch a property. Other properties include but are not limited toviability, motility, intact or damaged membranes, genetic defects, thepresence or absence of specific genes or gene markers, morphology andfertility. In certain embodiments, the user decides the property orproperties the device will analyze and select for, referred to as aproperty, or properties, of interest. For example, in one embodiment ofthe invention, if the property of interest is the presence of aY-bearing chromosome in sperm, the devices used with the invention cananalyze sperm and then select the sperm having the property of interestby, for example, photo-damaging/laser ablating the sperm having theproperty of interest. Alternatively, the devices used with the inventioncan photo-damage/laser ablate the sperm without the property ofinterest, leaving the sperm having the property of interest in tact. Inother embodiments, the devices of invention can select sperm having theproperty of interest by isolating the sperm having the property ofinterest, by for example, separating, or sorting, the sperm having theproperty of interest from sperm without the property of interest.Devices that may be used with the invention include but are not limitedto those disclosed above as well as in US Patent Application PublicationNo. US 2008/0153087 at paragraphs 23-87; paragraphs 99-148; and FIGS.1-5; and in U.S. Pat. No. 8,206,987 at column 28, line 17 to column 93,line 35; column 126, line 54 to column 130, line 3; and FIGS. 135-138;the disclosures of which are incorporated by reference herein.

Generally, sheath fluid is used to surround the stream of sample fluidas it travels through a flow cytometer or microfluidic chip.Furthermore, the sheath fluid may be introduced into a nozzle of a flowcytometer using pressurized gas or by a syringe pump. The pressurizedgas is often carbon dioxide or nitrogen. In certain embodiments of theinvention, a stream containing sperm to be analyzed may be comprised ofa sample fluid and a sheath fluid, or a sample fluid alone.

Optionally, the sample fluid or sheath fluid may also contain anadditive, such as, one or more OSRs, an antibiotic or a growth factor,as discussed above with respect to cell sample collection. Each of theseadditives may be added to either fluid in accordance therewith.

One embodiment of a sheath fluid comprisesTris(hydroxymethylaminomethane), sodium-citrate dihydrate and citricacid anhydrous. To prepare this low sugar sheath fluid, regardless ofthe intended volume of sheath fluid desired, the entire quantity of Trisand sodium-citrate are mixed with approximately 95% of the desiredvolume of ddH₂O. Three quarters of the citric acid is also concurrentlyadded. A titration with the remaining quantity of citric acid isperformed to reach a specific pH of 6.80. After pH adjustment, ddH₂O isadded until an osmolarity of 300 mOsm is obtained. One or more OSRs mayalso be added to this sheath fluid, typically in the range of 0.01 to5.0 mg/ml.

FIG. 1 illustrates, in schematic form, part of a flow cytometer used tosort a sperm composition to form one or more subpopulations, the flowcytometer being generally referenced as 10. In this particularembodiment of the invention, sex sorting is taking place so thesubpopulations are X-chromosome bearing sperm and Y-chromosome bearingsperm.

The flow cytometer 10 of FIG. 1 can be programmed by an operator togenerate two charged droplet streams, one containing X-chromosomebearing sperm, charged positively, 12, one containing Y-chromosomebearing sperm, charged negatively 13 while an uncharged undeflectedstream of dead cells 14 simply goes to waste.

An operator may also choose to program the flow cytometer in such amanner, that both the X- and Y-chromosome bearing sperm are collectedusing a “high purity sort” (in other words only live X- and Y-chromosomebearing sperm are collected) or to program the flow cytometer to collectboth the X- and Y-chromosome bearing sperm using an “enriched sort” (inother words it will collect droplets containing live cells that were notpreviously sorted and excluding all initial dead cells again by the useof Boolean Gate logic available with the computer that controls the flowcytometer). The Boolean Gate logic can also be used to collect only oneof either the X- or Y-chromosome bearing sperm.

Initially, a stream of sperm under pressure, is deposited into thenozzle 15 from the sperm source 11 in a manner such that they are ableto be coaxially surrounded by a sheath fluid supplied to the nozzle 15under pressure from a sheath fluid source 16. An oscillator 17 which maybe present can be very precisely controlled via an oscillator controlmechanism 18, creating pressure waves within the nozzle 15 which aretransmitted to the coaxially surrounded sperm stream as it leaves thenozzle orifice 19. As a result, the exiting coaxially surrounded spermstream 20 could eventually and regularly form droplets 21.

The charging of the respective droplet streams is made possible by thecell sensing system 22 which includes a laser 23 which illuminates thenozzle exiting stream 20, and the light emission of the fluorescingstream is detected by a sensor 24. The information received by thesensor 24 is fed to a sorter discrimination system 25 which very rapidlymakes the decision as to whether to charge a forming droplet and if sowhich charge to provide the forming drop and then charges the droplet 21accordingly.

A characteristic of X-chromosome bearing sperm is that they absorb morefluorochrome dye than Y-chromosome bearing sperm because of the presenceof more DNA, and as such, the amount of light emitted by the laserexcited absorbed dye in the X-chromosome bearing sperm differs from thatof the Y-chromosome bearing sperm and this difference communicates tothe sorter discrimination system 25 the type of charge to apply to theindividual droplets which theoretically contain only a single X- orY-chromosome bearing sperm. Dead cells (or those about to die) typicallyabsorb the quenching dye which is communicated to the sorterdiscrimination system 25 not to apply a charge to the dropletscontaining such cells.

The charged or uncharged droplet streams then pass between a pair ofelectrostatically charged plates 26, which cause them to be deflectedeither one way or the other or not at all depending on their charge intorespective collection vessels 28 and 29 to form respectively a genderenriched population of X-chromosome bearing and a gender enrichedY-chromosome bearing sperm having a DNA selective dye associated withtheir DNA. The uncharged non-deflected sub-population stream containingdead cells (or those about to die) go to the waste container 30.

Alternatively, the cells of a sperm composition may be sorted orenriched using laser steering. This is often referred to as opticaltrapping or holographic optical trapping. Generally, tightly focusedlaser light, such as, for example, light focused by a microscope lens,will have a steep intensity gradient. Optical traps use the gradientforces of a beam of light to trap particles based upon the dielectricconstant of the beam. To minimize its energy, a particle having adielectric constant greater than the surrounding medium will move to aregion of an optical trap where the electric field is highest. Suchdevices and methods are described, for example, in WO 2004/012133, U.S.Pat. No. 6,416,190 and related applications and patents, the content ofeach of which is hereby incorporated herein by reference. The cells ofthe sperm composition may be sorted accordingly into separatepopulations, wherein the spermatozoa of the populations comprises acertain percent X chromosome bearing or Y chromosome bearing sperm.Laser ablation/photo damage or fluid switching may also be used tocreate gender enriched populations.

Any of the steps of the cell sorting process may be carried out within atemperature range selected from the group consisting of about 5° C. toabout 15° C.; about 15° C. to about 20° C.; about 20° C. to about 25°C.; about 25° C. to about 30° C.; about 30° C. to about 35° C.; about35° C. to about 40° C. and about 40° C. to about 45° C.

Furthermore, it is contemplated that sorted or gender enriched sperm ofthe invention may comprise at least about 65% X chromosome bearing or Ychromosome bearing sperm, at least about 70% X chromosome bearing or Ychromosome bearing sperm, at least about 75% X chromosome bearing or Ychromosome bearing sperm, at least about 80% X chromosome bearing or Ychromosome bearing sperm, at least about 85% X chromosome bearing or Ychromosome bearing sperm, at least about 90% X chromosome bearing or Ychromosome bearing sperm, at least about 95% X chromosome bearing or Ychromosome bearing sperm, at least about 98% X chromosome bearing or Ychromosome bearing sperm, or at least about 99% X chromosome bearing orY chromosome bearing sperm.

C. Collection of the Sorted Cells into Catch Media

Once sorted, the sorted cells are collected in a vessel that contains acatch media. Generally, the purpose of the catch media includesproviding a fluid support for the cells. In one aspect of the invention,the catch media may comprise magnetic particles. Optionally, the catchmedia may further comprise any of the additives as discussed above withrespect to cell sample collection, including but not limited to one ormore OSRs, cryoprotectants, sterols, lipids, fatty acids and proteinsources. If included in the catch media, the sterols, lipids, and fattyacids may be, for example, cholesterol.

Exemplary protein sources include milk (including heat homogenized andskim), milk extract, egg yolk, egg yolk extract, soy protein and soyprotein extract. Such proteins may be used in a concentration from about1% (v/v) to about 30% (v/v), preferably from about 10% (v/v) to about20% (v/v), and more preferably about 10% (v/v).

Optionally, the catch media may also contain additives such as, anantibiotic, a growth factor or one or more OSRs, as discussed above withrespect to cell sample collection. Each of these additives may be addedto the catch media in accordance therewith.

One embodiment of a catch media is prepared by adding the appropriateamount of egg yolk to media comprising Tris(hydroxymethylaminomethane),sodium citrate dehydrate and citric acid anhydrous, mixing and thenallowing the mixture set overnight at 5° C. The following day themixture is centrifuged at 5000 g for 1.5 h. After centrifugation, thetop layer of the supernatant is discarded, and the remainingsupernatant, which comprises the low sugar catch media, is collected.One or more OSRs may also be added to the catch media, typically in therange of 0.01 to 5.0 mg/ml. The catch media should then be stored at 5°C. until use. Alternatively, the catch media can be frozen prior tostorage and/or shipping. It should be understood that magnetic particlesmay be added during any of the aforementioned steps for preparing catchmedia including prior to use, prior to freezing or subsequent to thawingof the media.

Gender enriched sperm may be collected into a vessel containing orcoated with a cryoextender comprising magnetic particles, acryoprotectant and optionally, any of the additives as discussed abovewith respect to sperm collection. In one embodiment, prior tocryoextension, storage or use of collected sperm, the magnetic particlesare removed from the catch media or the cryoextender prior to furtherprocessing or cryopreservation of the gender enriched sperm.

D. Cryopreservation of the Sorted Cells

Once the sperm have been sorted and caught in collection vessels, theymay be used for inseminating female mammals. This can occur almostimmediately, requiring little additional treatment of the sperm. In suchan instance, the sperm may be stored in their current state for a periodof time necessary to, for example, transport them to the location wherethe insemination is to take place. The sperm may, therefore, be storedand transported in, for example, the catch media.

Likewise, the sperm may be concentrated to a density appropriate for theparticular mammalian species, for example, a density of about 10×10⁶sperm/ml to about 120×10⁶ sperm/ml, in a magnetic particles andsubsequently stored and transported. The selected density depends uponfactors such as those discussed below with respect to fertilization,including the species of mammal from which the sperm were obtained. Sucha range of densities based upon the species of mammal from which thesperm were obtained are well known to those of skill in the art.

Likewise, the sperm may also be cooled or frozen for use at a laterdate. In such instances, the sperm may benefit from the addition of acryoextender to minimize the impact upon viability or post-thaw motilityas a result of cooling or freezing.

A protein source may be added to provide support to the sperm. Examplesof common protein sources include milk (including heat homogenized andskim), milk extract, egg yolk, egg yolk extract, soy protein and soyprotein extract. Such proteins may be found in a concentration fromabout 3% (v/v) to about 30% (v/v); from about 10% (v/v) to about 20%(v/v); or about 20% (v/v).

A cryoprotectant is preferably included in the cryoextender to lessen orprevent cold shock or to maintain fertility of the sperm. Numerouscryoprotectants are known in the art. Selection of a cryoprotectantsuitable for use with a given extender may vary, and depends upon thespecies from which the sperm to be frozen were obtained. Examples ofsuitable cryoprotectants include, for example, glycerol, dimethylsulfoxide, ethylene glycol, propylene glycol, trehalose, Triladyl®, andcombinations thereof. If included, generally, these cryoprotectants arepresent in the cryoextender in an amount, by percent volume, of about1-5%; 5%; about 5 to 10%; about 10%; about 10 to 20%; about 16.7%; about20%; about 20 to 30%; or about 30 to 40%.

Optionally, the cryoextender may also contain additives as discussedabove with respect to cell sample collection, including but not limitedto an antibiotic, a growth factor, or one or more OSRs. Each of theseadditives may be added to the cryoextender in accordance therewith.

The following method of freezing porcine sperm can be used with theinvention, but is presented by way of example only—any suitablecryopreservation method known in the art can be used.

After sorting, 50 ml tubes containing the sex sorted sperm (each with 20million cells) can be divided into tubes of 15 ml, with approximately 12ml of a sex-select sperm sample in each tube, each containingapproximately 10 million sex sorted sperm. Theses tubes can becentrifuged at 3076 g at 21° C. for 4 minutes. The supernatant decanted,and the pellet can remain with some of the supernatant in approximately50 μl.

To each pellet, a first cooling medium that may comprise a solution of20% egg yolk and/or lactose can then be added at room temperature. Themotility of the sperm can then be checked. If acceptable, the tubes canbe taken to a programmable temperature control machine(PolyScience—MiniTube) or can be manually handled to decrease thetemperature from about 21° C. to about 5° C. over a period of about 2hours. After the timed temperature shift with the cells at 5° C. afreezing medium is added to the cells, which may comprise egg-yolk,lactose, glycerol and EquexPasteStem, or may just comprise acryoprotectant such as glycerol, or the cryoprotectant with an osmoticstabilizer which is previously cooled to 5° C. is added to the samples.After 10 minutes, the sex sorted sperm composition can be placed inartificial insemination straws, and the straws then exposed to liquidnitrogen vapors (approximately 4 cm from the liquid nitrogen) for ashort period of time (e.g. 10 minutes) or cryopreserved in programmablefreezer and then placed directly into the liquid nitrogen for long termpreservation.

When gender enriched sperm compositions are ready for use, straws can beunfrozen by thawing/warming the straws (e.g. place in a water bath setat about 37° C. for about 1 minute or 500 for 20 seconds). Post-thaw,motility and viability of the sperm can then be analyzed at 30, 90and/or 150 minutes for standard comparisons. Magnetic particles can beadded to the thawed sperm composition and then subsequently removedprior to any further processing or use in AI.

III. Storage of the Collected Cells

A. Storage Period

Once the sperm have been collected from the source male, regardless ofwhether they are optionally sorted thereafter, the sperm may be storedfor a period of time. The period of storage is dependent upon severalfactors, including for example, the temperature at which the cells arestored, the number of cells within the storage container, whether thesperm are sorted or unsorted or have been previously been subjected toother processes such as cryopreservation, the method of fertilizationfor which the cells will be used, and the female mammal beingfertilized.

Generally, for example, the sperm may be stored for several hours, suchas for example, 2, 4, 8, 12, or 24 hours; for several days, such as forexample 1, 2, 3, 4, 5, 6, or 7 days; several weeks, such as for example,1, 2, 3, or 4 weeks; or several months, such as for example, 1, 2, or 3months. Typically, sperm may be stored for several hours to several daysat a temperature of about 0° C. to about 30° C.; for several days toseveral weeks at a temperature of about −4° C. to about 5° C.; and forseveral weeks to several months at a temperature of about −196° C. (inliquid nitrogen vapor) to about −4° C. For porcine sperm, for example, asample can be held at a temperature of 0-39° C. (typically 16-17° C.)for between about 12 hours to about 18 hours while it is being shippedfrom the collection point to the point of further processing or use. Inother embodiments of the invention, the sample can be held at atemperature of 0-39° C. (typically 16-17° C.) for more than 18 hours.

It is contemplated that in certain embodiments of the invention,magnetic particles are added to the storage medium and then subsequentlyremoved either before or after the storage period, e.g., prior to use inAI. Magnetic particles may be added and removed to a sperm compositionafter the storage period and prior to use as well.

B. Storage Temperature

The sperm, whether sorted or unsorted, may be stored at a range ofdifferent temperatures. Selection of a storage temperature is dependentupon several factors, such as for example, the length of time for whichthe sperm will be stored, the concentration of sperm within the storagecontainer, whether the sperm are sorted or unsorted, the method offertilization for which the sperm will be used, and the female beingfertilized. All of these factors affect the number of sperm that willremain viable during the storage period. By way of example, generallythe greater the length of time for which the sperm may be stored, thelower the temperature at which the sperm may be stored. In certainspecies, a decrease in temperature generally permits a greaterpercentage of the stored sperm to remain viable over a longer period oftime. In other species, such as with porcine sperm, this may not betrue.

Accordingly, sperm may be stored at a temperature of about −196° C. toabout 30° C. For example, sperm may be stored at a relatively lowstorage temperature, i.e., a temperature range of about −196° C. toabout −4° C.; in this embodiment, the temperature is typically fromabout −12° C. to about −4° C.; from about −10° C. to about −4° C.; orabout −4° C. Alternatively, the sperm may be stored at an intermediatestorage temperature, i.e., a temperature range of about −4° C. to about5° C.; in this embodiment, the temperature is typically at about −3° C.to about 5° C.; about 0° C. to about 5° C.; or about 5° C. In addition,the sperm may be stored at a moderately high storage temperature, i.e.,a temperature range of about 5° C. to about 30° C.; in this embodiment,the temperature is typically from about 10° C. to about 25° C.; fromabout 12° C. to about 23° C.; from about 15° C. to about 20° C.; orabout 18° C.

C. Storage Container

The sperm composition may be stored in a range of different containers.While the containers may vary in size, generally suitable containerswill be capable of containing the sperm composition; that is to say, thecontainers will be constructed of a material that is not susceptible toleaking or deterioration as a result of contact with fluids generally,and sperm compositions specifically, regardless of whether such contactoccurs on the inside or outside of the container. Examples of suitablecontainers include, for example, flasks, beakers, test tubes, ampules,and other such containers that are generally constructed of glass,plastic, or other similar materials. In a particular embodiment, thecontainer is of a type of construction that is used in the inseminationof a female, such as for example, an elongated container. Such elongatedcontainers may generally have a length to diameter ratio of about 1000:1to about 100:1; a length to diameter ratio of about 900:1 to about200:1; a length to diameter ratio of about 800:1 to about 300:1; alength to diameter ratio of about 700:1 to about 400:1; a length todiameter ratio of about 600:1 to about 400:1; a length to diameter ratioof about 500:1 to about 400:1; and in one particular embodiment, alength to diameter ratio of about 450:1. Such elongated containers maygenerally have a volume of 10 about 0.1 cc to about 100 cc, the volumeof the container selected to be used being based upon the species ofmammal from which the semen was collected. For example, the volume ofsuch elongated containers may be from about 0.1 cc to about 0.7 cc,preferably a volume of about 0.2 cc to about 15 0.6 cc, more preferablya volume of about 0.23 cc to about 0.5 cc, and most preferably a volumeof about 0.3 cc to about 0.4 cc.

In a particular embodiment, the elongated container is what is commonlyreferred to in the artificial insemination industry as a straw, having avolume of about 0.23 cc and a length to diameter ratio of about 133:1.In another particular embodiment, the elongated container is what iscommonly referred to in the artificial insemination industry as a straw,having a volume of about 0.5 cc and a length to diameter ratio of about67:1. Typically containers of these volumes are used for the storage ofbovine sperm.

Alternatively, the volume of the elongated containers may be from about1 cc to about 100 cc; about 10 cc to about 75 cc; about 15 cc to about50 cc; about 20 cc, to about 40 cc; or a volume of about 25 cc to about30 cc. In a particular embodiment, the elongated container is what iscommonly referred to in the artificial insemination industry as a straw,having a volume of about 25 cc and a length to diameter ratio of about445:1. Typically, containers of this volume are used for the storage ofporcine sperm.

The advantage of storing the sperm composition in a straw is that thecomposition may remain stored therein until it is to be used forinsemination of a female, at which time the contents of the straw may beplaced into the uterus of a female.

IV. Fertilization or Insemination

Another aspect of the present invention is the fertilization of an eggor insemination of a female, generally employing the novel process forprocessing and/or storing spermatozoa as described above.

Once a sperm composition has been formed as discussed in greater detailabove with respect to the collection and/or processing of a spermsample, the sperm composition may be used to inseminate a female.Insemination may be performed according to any of a number of ARTmethods well known to those of skill in the art. These methods include,for example, artificial insemination, including standard artificialinsemination, deep uterine insemination and laparoscopic insemination,and other methods well known to those of skill in the art. For example,a sperm composition comprising magnetic particles and one or more OSRsmay be used to inseminate a female, such as for example, by artificialinsemination. In a particular embodiment, the sperm composition may bein an elongated container for use in the insemination of a femalemammal.

Alternatively, the sperm composition may be used to fertilize an egg,and more particularly, an egg in vitro, such as for example, bymicroinjection, including intracytoplasmic sperm injection (ICSI), andother methods well known to those in the art. The fertilized egg maythereafter be introduced into the uterus of a female by any of a numberof means well known to those of skill in the art, such as for exampleembryo transplant. In another aspect of the invention, zygotes and/orembryos from artificially inseminated females can be recovered and thencultured and/or cryopreserved/vitrified.

Insemination of a female mammal or fertilization of an egg in vitro(followed by introduction of the fertilized egg into the uterus of afemale) using a sperm composition may occur shortly after formation ofthe sperm composition, such as for example, within about 120 hours;within about 96 hours; within about 72 hours; within about 48 hours, andin a particular embodiment, within about 24 hours after formation of thesperm composition. In such instances, generally the sperm compositionsmay not have been cryopreserved prior to insemination of a female orfertilization of an egg in vitro (i.e., the composition is fresh orcomprises fresh sperm) instead it may have been refrigerated attemperatures of about 4° C. to about 25° C.; about 10° C. to about 25°C.; about 15° C. to about 20° C.; or about 18° C. Alternatively, thesperm composition may be cryopreserved and then thawed prior toinsemination of a female or fertilization of an egg in vitro (i.e., thedispersion is frozen/thawed or comprises frozen/thawed sperm).Typically, in such an instance, the cryopreserved sperm composition willbe thawed immediately, such as, for example, within about 15 minutes,before insemination of a female or fertilization of an egg in vitro.Alternatively, the cryopreserved dispersion may be thawed over a periodof time or thawed and subsequently stored for a period of time, such asfor example less than about 5 days; less than about 2 days; less thanabout 1 day; or less than about 12 hours.

Example 1 Particle Preparation for Magnetic Removal of Dead/DamagedSperm

Magnetic Cores: Magnetic cores may be fabricated such as byco-precipitation of Fe₃O₄ with Fe₂O₃ so that the magnetic susceptibilityof the particles in a chosen magnetic field may be sufficiently high toprovide rapid separation of magnetically labeled cells from non-labeledcells. The core may be comprised of any magnetic material; possiblenon-limiting examples include: (1) ferrites such as magnetite, zincferrite, or manganese ferrite; (2) metals such as iron, nickel, orcobalt; and (3) chromium dioxide. In one embodiment, the iron cores arecomprised of magnetite (Fe₃O₄). In other embodiments the core may beextended to include substances such as other iron oxide basednanoparticle materials including composites having the general structureMFe₂O₄ (where M may be Co, Ni, Cu, Zn, Mn, Cr, Ti, Ba, Mg, or Pt).

Thus, in this one non-limiting example, a reaction chamber containing400 mL of dH₂O in a water kettle was warmed to 60° C. To the 400 mL ofwarmed dH₂O, 23.4 g of FeCl₃.6H₂O and 8.6 g of FeCl₂ or the like wasadded and the mixture was stirred under N₂ gas. To this solution, 30 mLof 25% NH₃.H₂O was added and mixing was continued under N₂ gas. Almostimmediately, the orange salt mixture turned to a dark brown/blacksolution. The heat was turned off and the ferrofluid slurry was allowedto cool while being vigorously stirred for 30 min. The precipitate wascollected magnetically and the supernatant was decanted. To themagnetically collected ferrofluid, 800 mL of dH₂O was added, swirled,and the magnetic collected process was repeated. The washing process wasrepeated four times to remove substantially all residual NH₃.H₂O and anynonmagnetic particles. The final wash step may include a solution of 800mL 0.02 M NaCl in dH₂O or the like. The collected iron core sizes werebetween approximately 3 and approximately 10 nm.

Coating of Iron Cores with a Functionalizable Surface: The final outerlayer may comprise a polymer coat that interacts with the aqueousenvironment and serves as an attachment site for proteins and ligands.Suitable polymers may include polysaccharides, alkylsilanes,biodegradable polymers such as, for example, poly(lactic acids) (PLA),polycaprolactone (PCL), and polyhydroxybutyrate-valerate (PHBV);composites, and polyolefins such as polyethylene in its differentvariants. More specifically, polysaccharide chains may include dextrans,arabinogalactan, pullulan, cellulose, cellobios, inulin, chitosan,alginates, and hyaluronic acid. Silicon containing compounds such asalkylsilanes may also be employed to encapsulate the magnetic core.Alkylsilanes suitable for embodiments of the described methods andprocesses, may include but are not limited to, n-octyltriethoxysilane,tetradecyltrimethoxysilane, hexadecyltriethoxysilane,hexadecyltrimethoxysilane, hexadecyltriacetoxysilane,methylhexadecyldiacetoxysilane, methyl-hexadecyldimethoxysilane,octadecyltrimethoxysilane, octadecyltrichlorosilane,octadecyltriethoxysilane, and 1,12-bis(trimethoxysilyl)dodecane. In oneexample, the ratio of iron to silicon containing compound coating may beapproximately 0.2. In other embodiments, the ratio of iron to siliconcontaining compound coating may be greater than about 0.2, such as about0.4 or 0.8, with a view toward completely coating the iron cores suchthat the iron cores may be removed from the cell suspension within themagnetic field. Undercoated particles may result in the free metal oxidecrystals which may be detrimental to cell viability. In still otherembodiments, the ratio of iron to silicon containing compound coatingmay be less than 0.2. Indeed, the iron concentration divided by thesilicon containing compound concentration may be from about 0.1 to about1.

For the examples of magnetic removal of dead/dying or compromised cellssuch as sperm, a silicon containing compound may be used to encapsulatethe iron cores.

The iron core precipitate may be allowed to settle. With theunderstanding that throughout this disclosure all amounts, times, andvalues may be varied up or down such as by 10%, 20%, 30%, or even 40% inany permutation or combination for some embodiments, 67.1 mg of theferrofluid were added to 100 mL of 10%2-(carbomethoxy)ethyltrimethoxysilane.2-(carbomethoxy)ethyltrimethoxysilane is yet another example of asilicon containing compound that is suitable for use in the describedmethods and processes as a silicon containing compound coating. The pHwas adjusted to 4.5 using >99.5% glacial acetic acid, and the suspensionwas reacted at 70° C. for 2 h under N₂ gas with vigorous mixing. Aftercooling, the particles may be magnetically collected and washed withdH₂O. After washing, the silane-coated magnetic nanoparticles may besuspended in 5 mL of 0.05 M 2-(N-morpholino)ethanesulfonic acid (MES)Buffer, TRIS Buffer, TALP buffer, or it may remain in the dH₂O until usefor separation. The resuspension buffer may be at a pH that retains orcreates a net negative zeta potential of the particles.

Iron concentration may be determined using Inductively CouplePlasma-Optical Electron Spectroscopy (ICP-OES), and the ironconcentration may be adjusted according to milligrams per milliliterneeded for optimal dead cell removal. The particles may have an averagehydrodynamic diameter of 300 nm, and need to be in a range of 300 to1000 nm to stay suspended in solution so that maximum interactionbetween the cells and particles is achieved by keeping the particles insuspension and not settling out due to larger sizes.

Coupling of proteins and ligands to the particle surfaces: In the eventthat the surface of the particles needs to be treated and conjugated toa protein or antibody, the following methods may be used. Periodatetreatment of dextran and other polymers are one method for theattachment of proteins due largely to the large number of reactivegroups that are available for modification. Mild sodium periodatetreatment may create reactive aldehyde groups by oxidation of adjacenthydroxyl groups or diols. Proteins, antibodies, streptavidin, andamino-modified nucleic acids may be added at high pH to allow amines toform Schiff bases with the aldehydes. The linkages may be subsequentlyreduced to stable secondary amine linkages by treatment with sodiumborohydride or sodium cyanoborohydride, which may reduce unreactedaldehyde groups to alcohols. Another method of coupling proteins to themagnetic nanoparticles may be to create stable hydrazine linkages. Forexample, a protein may be coupled to dextran using succinimidyl4-hydrazinonicotinate acetone hydrazone (SANH; Solulink Inc, San Diego,Calif.). The reaction may use five-fold less protein, and the resultingprotein density may appear as high as with other methods. The SANHreagent may allow more efficient and gentle coupling of ligands to thedextran surface. Ligand attachment on silica-coated magneticnanoparticles may be completed using (3-aminopropyl)triethoxysilane(APTS) to introduce amines on the particle surface while(3-mercaptopropyl)triethoxysilane (MPTMS) introduces SH groups. Theheterobifunctional coupling agent (Succinimidyl4-[N-maleimidomethyl]cyclohexane-1-carboxylate) may then be used to linkthiols to the amines. As examples, amines on the particle surface may belinked to thiols on streptavidin molecules, and thiols on the particlesurface may be linked to amines on streptavidin. There are severalmethods of crosslinking proteins through chemical modifications known inthe art that may be used for the present embodiments of the describedmethods and processes. For this example, the carboxylic acidfunctionalized silane may attach proteins and ligands through EDCchemistry.

EDC Activation of COOH groups on Particle Surface Activation: Thesilanized particles were re-suspended in 0.05 M MES buffer, collectedmagnetically, and the supernatant may be aspirated and discarded.Another 5 mL of MES buffer (0.05 M, pH 4.7-5.2) per 10 mg of iron wasadded to the particles and the suspension was vigorously shaken.Particles were magnetically collected, and the supernatant was aspiratedand discarded. This step may be repeated two or so additional times.Frozen EDC was allowed to thaw at room temperature for 30 min. EDC (alsoknown as EDAC or EDCI, 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide),commonly obtained as a hydrochloride, is a water soluble carbodiimide,which is typically employed at pH in the range between 4.0 and 6.0. EDCmay be used as a chemical crosslinker for collagen, reacting with thecarboxylic acid groups of the collagen polymer which may then bond tothe amino group in the reaction mixture.

1.6 mg of EDC/mg iron was added to the particle suspension and thesuspension was shaken vigorously. Each tube or the like containingparticles and EDC was be placed on a laboratory rocker at roomtemperature for 30 min. After 30 min., particles were magneticallycollected, and the supernatant was aspirated and discarded. Buffers ofvarying salt concentrations, molarities, including but not limited to,0.1 M to 1 M, and pH ranges from 10 to 4.7 may be used for proteinconjugation to the various surfaces. The function of each antibody,protein and ligand optimizes at different pH ranges and molarities, asis known in the art (Hermanson, Bioconjugate Techniques, 2008). In thisexample, the particle pellet was added to 0.05M MES buffer, particlesmay be magnetically collected, and supernatant may be aspirated anddiscarded. This step may be repeated three or so times. 10 mg ofprotein, ligand or stain was suspended in 0.05M MES buffer and added tothe particles so that the total labeling volume was about 5 mL per 10 mgof iron.

A stoichiometric balance of 1 mg of protein, ligand, or stain per 1 mgof iron was used for the coupling reaction. Some experiments suggestedthat the best binding of dead or compromised cells is concentrationdependent and may occur at about this concentration (with the abovepercentage variations applicable, of course). The range in antibody orprotein used may include, but is not limited to, 0.125 mg to 5 mg ofantibody per mg of iron. Tubes were shaken and placed on a laboratoryrocker at room temperature for 24 h, and particles were magneticallycollected. The supernatant was aspirated and discarded. Each particlesuspension was suspended in 5 mL of MES buffer. To each tube, 5 mL ofquenching solution (1M glycine, pH 8.0) was added and the tubes wereshaken vigorously. Quenching solutions may include, but are not limitedto, 2-mercaptoethanol, ethanolamine, glycine, UV exposure, sizeexclusion and magnetic collection. Each tube was placed on a laboratoryrocker for 30 min. at room temperature. After 30 minutes, 5 mL of washbuffer was added to each tube and shaken to mix. The particles weremagnetically separated, and the supernatant was aspirated and discarded.This step may be repeated 4 or so times. After the conjugation processwas complete, particles were magnetically collected, washed, andfiltered to obtain a size distribution of 50 to 400 nm. After the washsteps, each particle suspension was suspended in a buffer for theparticular cells, such as for sperm cells or other such cells, with a pH(6.0-8.0) and osmolarity (250-350) suitable for optimal sperm or othercell viability such as but not limited to TRIS solution, Sodium Citratesolution, TEST solution, egg yolk-TRIS (pH 6.5-7.4), egg yolk-sodiumcitrate (pH 6.5-7.4), egg yolk-TEST (pH 6.5-7.6), milk extenders (pH6.5-7.4), commercially available extenders marketed by IMVInternational, Maple Grove, Minn., USA and MiniTube GmbH, Vernona, Wis.,USA and chemically defied media including but limited to TALP (pH6.0-8.0) Tyrodes (pH 6.0-8.0) and Hepes BGM-3 (pH 6.0-8.0) so that theresultant working iron concentration was about 4 mg/mL as confirmed byInductive Coupled Plasma-Optical Emission Spectroscopy (ICP-OES).

The particles may be advantageously on the order of about 300 nm so thatinteraction between the particles and that of the damaged or dead cellsis maximized in solution. If particles are too large, such interactionmay not occur due to the settling effect of the larger sized particlesin solution. If particles are smaller than approximately 30 nm, they mayeither not be sufficiently magnetic and higher magnetic susceptibilitycore materials within a chosen magnetic energy field will have to begenerated, or these small particles may contribute to nonspecificbinding; that is, they may bind to viable cells as well as to dead anddying cells. If nonspecific binding relating to particle size isproblematic, particle size may either be increased, or a blocking agentdependent upon the particular cells involved, such as nonfat dried milkor serum albumin for sperm, may be added to the labeling buffer solutionto minimize such nonspecific binding.

The surface charged particles are comprised of Fe₃O₄ coated with2-(carbomethoxy)ethyltrimethoxysilane without further activation orfunctionalization. The ratio of iron to silicon containing compoundcoating is approximately 0.2.

Example 2 Removal of Dead Sperm

Three lots of magnetic particles were produced: Lot 1 was the firstbatch and used for field trial; Lot 2A was similar to Lot 1; Lot 2B wasmade using less silane per concentration of iron cores that Lot 1 andLot 2A; and Lot 3 was larger than Lot 2A and 2B combined and had aslightly larger load of iron per L. Also, Lot 3 was sonicated andcompared to Lot 3 without sonication.

All lots were sent for ICP and Zeta potential analysis. See FIG. 2 .

Lot 3 represents a 6 fold scale up of the step of production ofmagnetite cores and 150 fold scale up from the step of silane coating ofcores and magnetic recovery. The scale of Lot 3 produced ˜20 grams (ironcontent), which using 1 mg/160 million sperm results in sufficientparticles to treat ˜3000 billion sperm. Assuming an average ejaculate of10 billion sperm, approximately 300 ejaculates could be processed with20 grams of magnetic particles. Relatedly, since 50 semen straws can beproduced per billion sperm, ˜150,000 straws could be produced with 20grams of magnetic particles.

Lots 1, 2A, 2B and 3 were compared for dead removal capacity andPost-Thaw quality control data (e.g., motility, percent intact acrosomes[PIA] and progressive motility).

Procedure

-   1. Check volume, concentration, motility, morphology and pH of    ejaculates from 5 bulls. Add antibiotics.-   2. Extend the ejaculates 1:3 with TRIS media, pH 7.30.-   3. Centrifuge and adjust concentration to ˜1200 million sperm per    mL.-   4. Stain a 20 mL sample in modified TALP (20% yellow no. 6 [Y6] with    antioxidants), pH 7.4 at a concentration of 120 Million sperm    cells/mL with Hoechst 33342.-   5. Wash magnetic particles (2×) with modified TALP, pH 7.4, and    concentrate to 20 mg/mL.-   6. Divide sample in five 4 mL samples:    -   a. Control—keep as it is    -   b. Lot 1—add 1 mg of Lot 1 particles for every 160 million sperm    -   c. Lot 2A—add 1 mg of Lot 2A particles for every 160 million        sperm    -   d. Lot 2B—add 1 mg of Lot 2B particles for every 160 million        sperm    -   e. Lot 3—add 1 mg of Lot 3 particles for every 160 million sperm-   7. Incubate for 60 minutes at 34 C.-   8. Magnetically remove particles from treated samples.-   9. Check concentration of each treated sample.-   10. Place samples on the flow cytometer and record parameters. See    results in FIGS. 3 and 4 .-   10. Sex-sort 10 million cells into 3.5 mL of Tris A for each    treatment.-   11. Place in the cold room and cryopreserve samples.-   12. Perform post-thaw QC of each treatment (e.g., 0 and 3 Hour    Visual and IVOS Motility, membrane integrity via propidium iodide    (PI), and intact acrosomes via peanut agglutinin (PNA)). See results    in FIG. 5 .

Procedure

-   1. Check volume, concentration, motility, morphology and pH of    ejaculates from 5 bulls. Add antibiotics.-   2. Extend the ejaculates 1:3 with TRIS media, pH 7.30.-   3. Centrifuge and adjust concentration to ˜1200 million sperm per    mL.-   4. Stain 2.4×10⁹ sperm in 20 mL of modified TALP (20% Y6 with    antioxidants), pH 7.4 using Hoechst 33342.-   5. Wash magnetic particles (2×) with modified TALP, pH 7.4, and    concentrate to 20 mg/mL.-   6. Divide sample in five 4 mL samples:    -   a. Control—keep as it is    -   b. Lot 3 particles—add 1 mg of Lot 1 particles for every 160        million sperm    -   c. Lot 3 sonicated particles—add 1 mg of Lot 2A particles for        every 160 million sperm-   7. Incubate for 60 minutes at 34 C.-   8. Magnetically remove particles from treated samples.-   8. Check concentration of each treated sample-   9. Place samples on the flow cytometer and record parameters. See    results in FIGS. 6 and 7 .-   10. Sex-sort 10 million cells into 3.5 mL of Tris A for each    treatment.-   11. Place in the cold room and cryopreserve samples.-   12. Perform post-thaw QC of each treatment. See results in FIG. 8 .

Example 3 Use of Magnetic Particles During Staining Procedure

-   1. Collect ejaculates.-   2. Check volume, concentration, viability, motility, morphology and    pH. Add antibiotics.-   3. Standardize with TRIS media, pH 7.20, dilution 1:3.-   4. Centrifuge and concentrate to 1700-1800 million sperm/mL.-   5. Stain samples in modified TALP (20% Y6 with antioxidants), pH    7.4, at 160 million sperm per ml.-   6. Add 2 mg of particles/320 million sperm in modified TALP, pH 7.4.-   7. Incubate at 34° C. for 45 minutes.-   8. Magnetically collect particles for 2 minutes.    9. Sort on flow cytometer. See results in FIG. 9 .

Example 4 Removal of Dead Sperm in Modified TALP Procedure

-   1. Collect ejaculates from 4 bulls. Check volume, concentration,    viability, motility, morphology and pH. Add antibiotics.-   2. Stain ejaculates with Hoechst 33342 in modified TALP (20% Y6 with    antioxidants) (in 50 mL tubes), pH 7.4. The sperm concentration in    modified TALP will be 160 million sperm per mL.-   3. After 45 minutes of incubation at 34° C., divide samples in two:    -   A. 2 mL of treated sample (320 mill sperm total)    -   B. 2 mL of control sample (320 mill sperm total)-   4. Treated samples will be treated with 3.6 mg magnetic particles in    300 μL of modified TALP, pH 7.4.-   5. Control samples will be treated with the same μL of modified    TALP, pH 7.4 (without particles).-   6. Incubate samples 20 minutes at room temperature.-   7. Remove magnetic particles with magnet and place samples in 4 mL    tubes.-   8. Add 2 mL of red TALP to each sample.-   9. Check sperm concentrations in the samples (NucleoCounter—final    concentration of the control should be 80 million cells per mL).-   10. Place samples on the flow cytometer and record parameters.

Treatment with magnetic particles in modified TALP, pH 7.4, at 320million total sperm significantly removed dead cells in every bull(n=4). The average % of dead sperm removed according to the flowcytometer was 16%, taking samples from 20% to 4% dead. This increasedsort speed from 3800 to 4900 at an event rate of 20,000 events persecond (eps).

The average % sperm removed according to the NucleoCounter was 32%. 14%of cells were unaccounted for (when comparing NucleoCounter and flowcytometer data).

Example 5 Bulk-Sorting Sperm after Removal of Dead Sperm in ModifiedTALP Procedure

-   1. Collect ejaculates from 5 bulls. Check volume, concentration,    viability, motility, morphology and pH. Add antibiotics.-   2. Stain ejaculates with Hoechst 33342 in modified TALP (20% Y6 with    antioxidants) (in 50 mL tubes), pH 7.4. The sperm concentration in    modified TALP will be 160 million sperm per mL.-   3. After 45 minutes of incubation at 34° C., divide samples in two:    -   A. 2 mL of treated sample (320 mill sperm total)    -   B. 2 mL of control sample (320 mill sperm total)-   4. Treated samples will be treated with 3.6 mg magnetic particles in    300 μL of modified TALP, pH 7.4.-   5. Control samples will be treated with the same μL of modified    TALP, pH 7.4 (without particles).-   6. Incubate samples 20 minutes at room temperature.-   7. Remove magnetic particles with magnet and place samples in 4 mL    tubes.-   8. Add 2 mL of red TALP to each sample.-   9. Check sperm concentrations in the samples (NucleoCounter—final    concentration of the control should be 160 million cells per mL).-   10. Bulk sort 10 million cells per sample, check parameters,    cryopreserve and perform post-thaw QC. See results in FIGS. 10-14 .

Treatment with magnetic particles in modified TALP, pH 7.4, at 320million total sperm increased sort rate from 2600 to 3200 at an eventrate of 20,000 eps.

Example 6 Sex-Sorting Sperm after Removal of Dead Sperm in Modified TALPProcedure

-   1. Collect ejaculates from 8 bulls. Check volume, concentration,    viability, motility, morphology and pH. Add antibiotics.-   2. Stain ejaculates with Hoechst 33342 in modified TALP (20% Y6 with    antioxidants), pH 7.4.-   3. Place 1 mL of the stained sample in a 50 mL tube and treat with    magnetic particles in modified TALP, pH 7.4.-   4. Place 1 mL from the stained sample in a 4 mL tube and treat it    with 250 μL of modified TALP, pH 7.4, without magnetic particles.-   5. Incubate at room temperature for 20 min.-   6. Remove magnetic particles with magnet and place treated sample in    a 4 mL tube.-   7. Check concentration of control and treated sample in    NucleoCounter.-   8. Add 1 mL of red TALP to each sample.-   9. Sex-sort 10 million sperm for control, 10 million for treated    sample, check parameters, cryopreserve and perform post-thaw QC. See    results in FIGS. 15-18 .

Example 7 Removal of Dead Sperm with Magnetic Particles and IVFProcedure

-   1. Collect ejaculates from 8 bulls. Check volume, concentration,    viability, motility, morphology and pH. Add antibiotics.-   2. Staining volume in modified TALP (20% Y6 with antioxidants), pH    7.4 and Hoechst 33342 will be 4 mL. After 45 minutes of incubation    at 34° C., samples divided in two: one will be used as control and    the other will be treated with magnetic particles (each one will a    total of 320 million sperm in 2 mL).-   3. 3.6 mg of particles in water, collected with the magnet and    re-suspended in 600 μL of modified TALP, pH 7.4, will be used for    each sample at a concentration of 320 million sperm.-   4. After adding the particles, the samples will be incubated 20    minutes at room temperature. Control samples will be kept in the    same conditions but without the particles.-   5. After treatment with magnetic particles, sperm concentrations    will be determined in the treated and the control samples to    quantify the % of total sperm (dead+unaccounted) removed by the    particles and 2 mL of red TALP will be added for dead sperm    quenching. Data from individual sorter histograms (using production    sorters) will provide results for the amount of dead sperm (with and    without treatment) as well as flow cytometer performance. See    results in FIGS. 20-22 .-   6. Samples will be sex-sorted, frozen (5 straws per treatment for    each bull) and sent to IVF trials. Standard post-thaw QC analysis    will also be performed. See results in FIGS. 23 and 24 .

Example 8 Production of Magnetic Particles Procedure

Production of Cores:

-   1. Measure out dH₂O and place in glass beaker (200 mL).-   2. Weight out FeCl₃.6H₂O (11.68 grams).-   3. Weigh out FeCl₂.4H₂O (4.30 grams)-   4. Dissolve iron salts separately in cold water (use ⅓ of the total    volume of H₂O to dissolve each salt).-   5. Mix both salts.-   6. Use the remaining water to recuperate as much iron as possible    from the beakers where the salts were dissolved.-   7. Insert nitrogen source via tubing into the H₂O. Turn on nitrogen    taking care not to exceed ˜15 psi so as not to cause splashing.-   8. Insert Mixer and start mixing the salts slowly.-   9. Turn on the water kettle or water bath—Target temperature is 85°    C.-   10. Allow 15 minutes for appropriate mixing of the iron salts.-   11. Add 25% NH₃.H₂O slowly to the iron salt mixture (30 mLs).-   12. Turn heat off and maintain mixing for 15 minutes.-   13. Allow ferrofluid to cool to room temperature.-   14. Place on glass beaker and place in magnetic holder. After    ferrofluid has collected, decant off supernatant.-   15. Repeat ferrofluid magnetic collection and wash with dH₂O—3    times.-   16. After the final dH₂O wash has been completed, wash ferrofluid    with 0.02 M NaCl.-   17. Once wash with 0.02 M NaCl is complete, magnetically collect    ferrofluid again and resuspend in dH₂O.-   18. Dehydrate 10 mL of particles, weight and calculate the amount of    iron and the dilution in water to achieve a concentration of 33.0    mg/mL.    Silane coating of Cores:-   1. Ferrofluid (67.6 mgs) is added to 10% Carboxyethylsilanetriol    sodium salt in water (35.5 mLs).-   2. The pH is adjusted with >99% glacial acetic acid so that the pH    reads 4.5.-   3. The suspension is reacted at 70° C. for 2 hours with mixing    (evaporate ˜½ of the volume).-   4. Allow coated particles to cool. Magnetically collect them in    glass beaker and decant the supernatant.-   5. Wash the particles with water and magnetically collect    again—repeat 4 times.-   6. After washes, the silane coated magnetic nanoparticles are    resuspended in storage buffer (pH 7.4) at a known volume.-   7. A sample is sent out for ICP analysis, and the resultant iron    measurement dictates the resuspension volume of iron (usually 4    mg/mL).

Example 9

A batch of iron cores was produced (1 L, 58.4 grams of FeCl₃.6H₂O and21.5 grams of FeCl₂.4H₂O).

After the reaction of the iron salts (using 25% NH₃.H₂O at 85° C.), thecores were washed in water and 0.02M NaCl in a volume of ˜250 mL. Coreswere kept in water at 4° C. Iron concentration in the ferrofluidmeasured 14.76 mg/mL of iron (53% yield).

67.6 mg of the ferrofluid was used for the production of coated magneticparticles. After the reaction with 10% carboxyethylsilanetriol sodiumsalt, the particles were washed in water and kept in bead storagebuffer. Analyzed iron concentration using ICP measured 3.342 mg/ml. Zetapotential was measured to make sure that all the magnetic particles wereproperly coated. See FIG. 25 .

Example 10 Titration of Magnetic Particles Procedure

-   1. Obtain ejaculates from 15 bulls.-   2. Treat with TALP medium, pH 7.3 (1:3 dilution).-   3. Stain samples in modified TALP (20% Y6 with antioxidants), pH    7.4, at a 160 million sperm per mL using Hoechst 33342.-   4. Incubate 45 minutes.-   5. Divide sample in 5 aliquots:    -   Control    -   3.5 mg of particles for every 320 million sperm    -   3.0 mg of particles (in 200 μL modified TALP, pH 7.4) for every        320 million sperm    -   2.5 mg of particles (in 200 μL modified TALP, pH 7.4) for every        320 million sperm    -   2.0 mg of particles (in 200 μL modified TALP, pH 7.4) for every        320 million sperm    -   1.0 mg of particles (in 200 μL modified TALP, pH 7.4) for every        320 million sperm-   6. Add 300 μL of modified TALP, pH 7.4, to the control sample to    keep the volumes equal.-   7. Incubate samples for 20 minutes at room temperature.-   8. Check concentration on NucleoCounter.-   9. Place samples on the flow cytometer and record parameters. See    results in FIGS. 26 and 27 .

Example 11 Magnetic Particles Used in Live and Dead Sperm Mixed SamplesProcedure

-   1. Take a good quality neat ejaculate (total of 10 ejaculates).-   2. Dilute ejaculate with 3× the amount of TALP medium, pH 7.3.-   3. Centrifuge and resuspend to 1600 million sperm per mL.-   4. Divide treated ejaculate in two parts: A and B.-   5. Place part A in the cabinet and part B in the freezer for 1 hour.-   6. Thaw part B.-   7. Mix parts A and B in the following proportions:    -   Only A (low % dead sample)    -   3A:1B    -   2A:2B    -   1A:3B (very high % dead sample)-   8. Stain each mix at 160 million sperm per mL in modified TALP (20%    Y6 with antioxidants), pH 7.4 (2 mL samples) with Hoechst 33342.    Incubate 45 minutes.-   9. Add treatment with magnetic particles (2 mg per 320 million).-   10. Incubate 20 minutes.-   11. Magnetically collect particles. Place modified TALP, pH 7.4,    with live sperm in a 4 mL tube.-   12. Check concentration (NucleoCounter).-   13. Place samples on the flow cytometer and record parameters. See    results in FIG. 28 .

Example 12 Magnetic Particles Used Before/During Staining in ModifiedTALP Procedure

-   1. Take 6 neat ejaculates.-   2. Dilute ejaculates with 3× the amount of TALP medium, pH 7.3.-   3. Centrifuge and resuspend to 1800 million sperm per mL.-   4. Stain in modified TALP (20% Y6 with antioxidants), pH 7.4, at 160    million sperm per mL (9 mL samples) in Hoechst 33342.-   5. Divide 9 mL in 3 aliquotes of 3 mL:    -   A. Control    -   B. Particles before incubation    -   C. Particles during incubation    -   D. Particles after incubation-   6. Add magnetic particles (2 mg/360 million sperm) only to sample B.-   7. Incubate for 25 minutes.-   8. Take samples out of the water bath and add magnetic particles (2    mg/360 million sperm) only to sample C.-   9. Place into incubator for another 20 minutes.-   10. Take samples out of the water bath and add magnetic particles (2    mg/360 million sperm) only to sample D (incubate for 20 minutes).-   11. Add the same volume of modified TALP, pH 7.4, without particles    to sample A.-   12. Magnetically collect particles in samples B, C and D. Place    modified TALP, pH 7.4, with live sperm in a 4 mL tube.-   13. Check concentration (NucleoCounter).-   14. Place samples on the flow cytometer and record parameters. See    results in FIGS. 29 and 30 .

Example 13 Magnetic Particles Used Before Staining in Modified TALPProcedure

-   1. Get 5 fresh ejaculates.-   2. Check volume, concentration, motility, morphology and pH. Add    antibiotics.-   3. Standardize with TALP medium, pH 7.2 (dilution 1:3).-   4. Centrifuge and concentrate to 1700-1800 million sperm/mL.-   5. Dilute each sample to 160 million sperm per mL in a 4 mL final    volume in modified TALP (20% Y6 with antioxidants), pH 7.4.-   6. Divide each sample in 2: Control and Treatment.-   7. Add magnetic particles to the “Treatment” sample (2 mg/320    million sperm) and equal volume of modified TALP to the “Control”    sample.-   8. Incubate for 10 minutes.-   9. Add Hoechst 33342 stain to all the samples (17 μL per 320 million    sperm).-   10. Incubate 45 minutes at 34° C.-   11. Magnetically collect the particles in the treated sample.-   12. Place samples on the flow cytometer and record parameters. See    results in FIGS. 31 and 32 .

Example 14 Magnetic Particles Recovery After Second Wash Procedure

-   1. Get 5 fresh ejaculates.-   2. Treat with TALP medium, pH 7.3 (1:3 dilution).-   3. Stain a 4 mL sample in modified TALP (20% Y6 with antioxidants),    pH 7.4, at a 160 million sperm per mL.-   4. Divide sample in 2 aliquots (2 mL each): Control and Treated-   5. Add magnetic particles (2 mg/320 million sperm) to Treated sample    and equivalent volume of modified TALP, pH 7.4, to Control sample.-   6. Incubate 45 minutes. Collect magnetic particles.-   7. Check concentration.-   8. Place samples on the flow cytometer and record parameters.-   9. Analyze viability on control sample, treated sample and magnetic    particles (resuspended in 300 μL of modified TALP, pH 7.4): Take 50    μL each sample and dilute into 1 mL of modified TALP, pH 7.4. Add 15    μL of SYBR® and incubate 10 minutes at 34° C. Add 5 μL of PI. Check    viability on analyzer. See results in FIGS. 33 and 34 .

Example 15 IVF Procedure

-   1. Get 5 fresh ejaculates.-   2. Check volume, concentration, motility, morphology and pH. Add    antibiotics.-   3. Standardize with TALP medium, pH 7.2 (dilution 1:3).-   4. Centrifuge and concentrate to 1700-1800 million sperm/mL.-   5. Stain a 6 mL sample for each bull in modified TALP (20% Y6 with    antioxidants), pH 7.4, at 160 million sperm per mL with Hoechst    33342.-   6. Divide each sample in 2 tubes (4 mL tubes filled with 3 mL of    sample):    -   Control    -   Treated-   7. Treat sample B with 0.387 μL of pre-washed particles (=2 mg/320    million cells) and sample A with 0.387 μL of modified TALP, pH 7.4.-   8. Incubate at 34° C. for—45 minutes.-   9. Collect particles.-   10. Check concentration on NucleoCounter.-   11. Sort 15 million cells on flow cytometer and record parameters.    See result for 2 sets in FIGS. 35, 36 and 38-40 .-   12. Place in the cold-room for 90 minutes and cryopreserve 4-5    straws (2.1 million cells per 1%4 cc straw) in TRIS AB.-   13. Check post-thaw QC: Motility (0 and 3 hour), SYBR®/PI Viability    and Purity. See results for 2 sets in FIGS. 37 and 41 .-   14. Conduct IVF trials. See results in FIG. 42 .

Example 16 Field Trial

Ejaculates from 5 bulls were divided and treated with magnetic particlesduring staining with Hoechst 33342 and then sex-sorted on a flowcytometer after removal of the magnetic particles. 637 heifers and 731cows were then inseminated with either control samples (sex sorted, notreatment with magnetic particles) or samples treated with magneticparticles. The pregnancy rate for the control group was 44.7% and forthe treatment group was 43.9%. The conception rates for control cows was32.94% (SE=3.7%), for control heifers 50.93% (SE=2.68%), for treatmentcows 39.30% (SE=2.42%) and for treatment heifers 49.17% (SE=3.24%).

As can be easily understood from the foregoing, the basic concepts ofthe present invention may be embodied in a variety of ways. Theinvention involves numerous and varied embodiments using sex sortedsperm to increase the genetic progress of a line, including, but notlimited to, the best mode of the invention.

As such, the particular embodiments or elements of the inventiondisclosed by the description or shown in the figures or tablesaccompanying this application are not intended to be limiting, butrather exemplary of the numerous and varied embodiments genericallyencompassed by the invention or equivalents encompassed with respect toany particular element thereof. In addition, the specific description ofa single embodiment or element of the invention may not explicitlydescribe all embodiments or elements possible; many alternatives areimplicitly disclosed by the description and figures.

The invention claimed is:
 1. A method of processing sperm cellscomprising the steps of: a) forming a stream comprising the sperm cells;b) determining whether a property of interest is present or absent inthe sperm cells in the stream; c) ablating or photodamaging asubpopulation of the sperm cells based on the presence or absence of theproperty of interest in the sperm cells; d) collecting the sperm cells;e) adding magnetic particles to the collected sperm cells to form amixture, the magnetic particles having a diameter of 30 to 1000 nm andcomprising a chargeable silicon-containing compound, the magneticparticles binding to the ablated or photodamaged subpopulation of spermcells solely through an electrical charge interaction; f) applying amagnetic field to the magnetic particle-bound sperm cells; and g)removing the magnetic particle-bound subpopulation from the mixture. 2.The method of claim 1, the magnetic particles comprising an outer layercomprising a polysaccharide, an alkylsilane, a polylactic acid, apolycaprolactone, polyhydroxybutyrate-valerate or a polyolefin.
 3. Themethod of claim 2, the outer layer further comprising a chargeable, orcharged, moiety.
 4. The method of claim 3, the chargeable, or charged,moiety comprising a metal, an oxide, a carboxylate, an amine, an amide,a carbamide, a sulfate, a sulfonate, a sulfite, a phosphonate, aphosphate, a halide or a hydroxide.
 5. The method of claim 1, whereinthe mixture has a pH of 6.5 to 8.0.
 6. The method of claim 1, whereinthe mixture comprises a buffer.
 7. The method of claim 1, wherein themixture comprises sperm cells at a concentration of 120×10⁶ cells/ml to500×10⁶ cells/ml.
 8. The method of claim 1, wherein the magneticparticles are non-toxic to the sperm cells.